Compositions and methods for identifying enzyme and transport protein inhibitors

ABSTRACT

The invention is directed to compositions to screen for small molecule drugs that inhibit proteases, such as viral proteases, e.g., HIV proteases; and methods for making and using these compositions. The invention provides compositions and methods for identifying compositions, e.g., drug molecules, that can inhibit proteases, e.g., HIV proteases. In alternative embodiments, the invention provides cell-based assays to screen for compositions, e.g., small molecules or drugs, that inhibit or modify the activity of enzymes such as calcium-dependent protein convertases involved in HIV envelop protein processing, including cleavage of the HIV gp160 envelope precursor, resulting in gp120 and gp41 envelope products.

TECHNICAL FIELD

This invention relates to molecular and cellular biology, biochemistry,molecular genetics, and drug design and discovery. In one aspect, theinvention is directed to compositions to screen for small molecule drugsthat inhibit viral proteases.

BACKGROUND

Current treatments for human immunodeficiency virus (HIV) includeinhibitors of HIV proteases; but these inhibitors can have severe sideeffects. Also, there has been a rapid emergence of HIV strains that aredrug resistant, e.g., insensitive to currently used HIV inhibitors,including HIV protease inhibitors.

HIV-1 protease (PR), an aspartyl protease, is required for the efficientprocessing of the Gag and Gag-Pol precursor polyproteins; a criticalstep in the viral life cycle. For this reason, targeting PR has longbeen the focus of anti-retroviral therapy. However, aside from itsproteolytic activity, its effects on the host cell are still unclear.Cytotoxic effects, together with instability, render expression of PR inmammalian cells difficult. Elucidating the role of PR in the viral lifecycle, as well as discerning its effects on the host machinery, is vitalfor the design of novel therapeutic approaches.

SUMMARY

The invention provides cell-based methods for monitoring the activity ofa protease, viral protease or an HIV-1 protease (PR) comprising:

(1) (a) providing a nucleic acid encoding a scaffold protein operativelylinked to a transcriptional regulatory unit, wherein the scaffoldprotein comprises:

-   -   (i) an amino acid motif or subsequence susceptible to cleavage        by the protease, viral protease or HIV-1 protease (PR) under        physiologic (cell culture) conditions;    -   (ii) a transmembrane domain;    -   (iii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iv) a detectable moiety,    -   wherein the amino acid motif or subsequence susceptible to        cleavage by the protease, viral protease or HIV-1 protease (PR)        is positioned within the scaffold protein such that when the        detectable moiety is cleavage away from (off from) the scaffold        protein by the protease, viral protease or HIV-1 protease (PR)        the remaining subsequence of scaffold protein on the        extracellular surface of the cell lacks the detectable moiety;

(b) providing a nucleic acid encoding the protease, viral protease orHIV-1 protease (PR) operatively linked to a transcriptional regulatoryunit, or a cell that expresses a heterologous or endogenous protease,viral protease or HIV-1 protease (PR);

(c) inserting (transfecting) the nucleic acid of the invention into thecell if the cell does not already express a heterologous or endogenousprotease, viral protease or HIV-1 protease (PR);

(d) co-expressing the nucleic acid of the invention in the cell, orexpressing the nucleic acid of (a) in the cell if the cell alreadyexpresses a heterologous or endogenous protease, viral protease or HIV-1protease (PR); and

(e) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell,

wherein an intact scaffold protein comprising the detectable moiety isexpressed on the extracellular surface of the cell when the protease,viral protease or HIV-1 protease (PR) is not enzymatically active, andan intact scaffold protein is not or is substantially less expressed onthe extracellular surface of the cell when the protease, viral proteaseor HIV-1 protease (PR) is enzymatically active (the detectable moiety iscleaved off by the protease, viral protease or HIV-1 protease (PR));

(2) the method of (1), wherein the scaffold protein further comprises anendoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1)motif,

wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positionedin the scaffold protein such that when the protease is active thescaffold will be separated into two pieces, leaving the ER retentionmotif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of thepolypeptide in the ER and freeing the detectable moiety-comprisingportion to the cell's extracellular membrane, and if the protease isblocked or inactive, the entire scaffold polypeptide will be retained inthe ER, and as a consequence will not be detected on the cell'sextracellular surface; or

(3) the method of (1), wherein the scaffold protein further comprises ap2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold;wherein optionally the p2/p7 recognition sequence comprises or consistsof ATIMMQRGN (SEQ ID NO:2), or optionally an exemplary amino-acidsequence of p2/p7 comprisesAEAMSQVTNS/ATIMMQRGN/FRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWK CGKEGHQMKDCTERQANATIMMQRGN (SEQ ID NO:3).

In alternative embodiments the methods of the invention further comprisescreening for an inhibitor of an HIV-1 protease (PR) by:

(a) providing a compound to be screened as an inhibitor of a protease,viral protease or HIV-1 protease (PR), or providing a nucleic acid to bescreened as encoding an inhibitor of a protease, viral protease or HIV-1protease (PR);

(b) contacting a plurality of the cells with the compound or nucleicacid of (a) either before, during and/or after the co-expressing thenucleic acid in the cell; and

(c) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell,

wherein an intact scaffold protein comprising the detectable moiety isexpressed on the extracellular surface of the cell when the protease,viral protease or HIV-1 protease (PR) is inhibited by: the compound, acomposition encoded by the nucleic acid, or a compound present in thecell only because the nucleic acid was expressed, and an intact scaffoldprotein is not or is substantially less expressed on the extracellularsurface of the cell when the protease, viral protease or HIV-1 protease(PR) is enzymatically active (the detectable moiety is cleaved off bythe protease, viral protease or HIV-1 protease (PR)) and the enzymaticactivity of the protease, viral protease or HIV-1 protease (PR) is notsignificantly inhibited by: the compound, a composition encoded by thenucleic acid, or a compound present in the cell only because the nucleicacid was expressed.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ofthe invention in the cell and not adding the compound to be screened asan inhibitor to one of the divided cell samples.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ofthe invention in the cell and adding a known inhibitor of the HIV-1protease (PR) to one of the divided cell samples.

In alternative embodiments, the amino acid motif or subsequencesusceptible to cleavage by the protease, viral protease or HIV-1protease (PR) under physiologic (cell culture) conditions comprisesLEAQEEEEVGF (SEQ ID NO:3), or ATIMMQRGN (SEQ ID NO:2), or comprises amotif defined by and/or is definable by a protocol as described by Kurt(2003) Biophys J. 85(2):853-63; or Prabu-Jeyabalan (2002) Structure10(3):369-81; or Prabu-Jeyabalan (2006) J. Virol. 80(7):3607-16; orKontijevskis (2007) Proteins 68(1):305-12; or Shoeman (1991) FEBS Lett.278(2):199-203.

The HIV-1 protease (PR) can comprise or consist of SEQ ID NO:4 or SEQ IDNO:5, or an enzymatically active fragment thereof:

(SEQ ID NO: 4) PQVTLWQRPL VTIKIGGQLK EALLDTGADD TVLEEMSLPGRWKPKMIGGI GGFIKVRQYD QILIEICGHKAIGTVLVGPT PVNIIGRNLL TQIGCTLNF(SEQ ID NO: 5) PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF

The exemplary SEQ ID NO:5 is a prototype of an NL4-3 genome, but can beused with any other sequences from other strains of HIV.

In alternative embodiments the transcriptional regulatory unit comprisesa promoter, an inducible promoter or a constitutive promoter.

In alternative embodiments the cell is a mammalian cell, a monkey cellor a human cell.

In alternative embodiments the scaffold proteins of the inventioncomprise all or part of a mouse Lyt2 or a human CD8 polypeptide.

In alternative embodiments the detectable moiety comprises an epitopefor an antibody, or a FLAG tag. The detectable moiety can be detected ormeasured on the extracellular surface of the cell by a high throughputscreen, a flow cytometry or microscope visualization.

In alternative embodiments the compound to be screened as an inhibitorof the protease, viral protease or HIV-1 protease (PR) comprises a smallmolecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, apolysaccharide or a lipid.

In alternative embodiments the compound to be screened as an inhibitorof the protease, viral protease or HIV-1 protease (PR) is a member of alibrary of compounds to be screened, or a member of a random peptidelibrary or a chemical compound library.

The invention provides cell-based methods for monitoring the activity ofa protease comprising:

(1) (a) providing a nucleic acid encoding a scaffold protein operativelylinked to a transcriptional regulatory unit, wherein the scaffoldprotein comprises:

-   -   (i) an amino acid motif or subsequence susceptible to cleavage        by the protease under physiologic (cell culture) conditions;    -   (ii) a transmembrane domain;    -   (iii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iv) a detectable moiety,    -   wherein the amino acid motif or subsequence susceptible to        cleavage by the protease is positioned within the scaffold        protein such that when the detectable moiety is cleavage away        from (off from) the scaffold protein by the protease the        remaining subsequence of scaffold protein on the extracellular        surface of the cell lacks the detectable moiety;

(b) providing a nucleic acid encoding the protease operatively linked toa transcriptional regulatory unit, or a cell that expresses aheterologous or endogenous protease;

(c) inserting (transfecting) the nucleic acid of the invention into thecell if the cell does not already express a heterologous or endogenousprotease;

(d) co-expressing the nucleic acid of the invention in the cell, orexpressing the nucleic acid of (a) in the cell if the cell alreadyexpresses a heterologous or endogenous protease; and

(e) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell,

wherein an intact scaffold protein comprising the detectable moiety isexpressed on the extracellular surface of the cell when the protease isnot enzymatically active, and an intact scaffold protein is not or issubstantially less expressed on the extracellular surface of the cellwhen the protease is enzymatically active (the detectable moiety iscleaved off by the protease); or

(2) the method of (1), wherein the scaffold protein further comprises anendoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1)motif,

wherein the ER retention motif or KDEL motif is positioned in thescaffold protein such that when PR is active the scaffold will beseparated into two pieces, leaving the ER retention motif-comprising orKDEL motif-comprising portion of the polypeptide in the ER and freeingthe detectable moiety-comprising portion to the cell's extracellularmembrane, and if PR is blocked or inactive, the entire scaffoldpolypeptide will be retained in the ER, and as a consequence will not bedetected on the cell's extracellular surface.

In alternative embodiments the methods of the invention further comprisescreening for an inhibitor of a protease by:

(a) providing a compound to be screened as an inhibitor of protease, orproviding a nucleic acid to be screened as encoding an inhibitor ofprotease;

(b) contacting a plurality of the cells with the compound or nucleicacid of either before, during and/or after the co-expressing the nucleicacid in the cell; and

(c) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell,

wherein an intact scaffold protein comprising the detectable moiety isexpressed on the extracellular surface of the cell when the protease isinhibited by: the compound, a composition encoded by the nucleic acid,or a compound present in the cell only because the nucleic acid wasexpressed, and an intact scaffold protein is not or is substantiallyless expressed on the extracellular surface of the cell when theprotease is enzymatically active (the detectable moiety is cleaved offby the protease) and the enzymatic activity of the protease is notsignificantly inhibited by: the compound, a composition encoded by thenucleic acid, or a compound present in the cell only because the nucleicacid was expressed.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ofthe invention in the cell and not adding the compound to be screened asan inhibitor to one of the divided cell samples.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ofthe invention in the cell and adding a known inhibitor of the proteaseto one of the divided cell samples.

In alternative embodiments the transcriptional regulatory unit comprisesa promoter, or an inducible promoter, or a constitutive promoter.

In alternative embodiments the cell is a mammalian cell, a monkey cellor a human cell.

In alternative embodiments the scaffold proteins of the inventioncomprise all or part of a mouse Lyt2 or a human CD8 polypeptide.

In alternative embodiments the detectable moiety comprises an epitopefor an antibody, or a FLAG tag. The detectable moiety can be detected ormeasured on the extracellular surface of the cell by a high throughputscreen, a flow cytometry or microscope visualization.

In alternative embodiments the compound to be screened as an inhibitorof protease comprises a small molecule, a nucleic acid, a polypeptide orpeptide, a peptidomimetic, a polysaccharide or a lipid. The compound tobe screened can be an inhibitor of protease is a member of a library ofcompounds to be screened, or a member of a random peptide library or achemical compound library.

In alternative embodiments the protease is an HIV-1 protease (PR), orthe protease is a viral, a microbial or a mammalian protease.

The invention provides cell-based methods for monitoring the activity ofa cell's ER and/or trans-Golgi network comprising:

(1) (a) providing a nucleic acid encoding a scaffold protein operativelylinked to a transcriptional regulatory unit, wherein the scaffoldprotein comprises:

-   -   (i) a transmembrane domain;    -   (ii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iii) a detectable moiety;

(b) inserting (transfecting) the scaffold protein-encoding nucleic acidof (a) into the cell;

(d) expressing the nucleic acid of (a); and

(e) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell,

wherein the scaffold protein is expressed on the extracellular surfaceof the cell when the activity of the cell's ER and trans-Golgi networkis functioning; or

(2) the method of (1), wherein the scaffold protein further comprises anendoplasmic reticulum (ER) retention motif or a KDEL motif,

wherein the ER retention motif or KDEL motif is positioned in thescaffold protein such that when PR is active the scaffold will beseparated into two pieces, leaving the ER retention motif-comprising orKDEL motif-comprising portion of the polypeptide in the ER and freeingthe detectable moiety-comprising portion to the cell's extracellularmembrane, and if PR is blocked or inactive, the entire scaffoldpolypeptide will be retained in the ER, and as a consequence will not bedetected on the cell's extracellular surface.

In alternative embodiments the methods of the invention further comprisescreening for an inhibitor of the cell's ER and trans-Golgi network by:

(a) providing a compound or nucleic acid to be screened as an inhibitorof the cell's ER and trans-Golgi network;

(b) contacting a plurality of the cells with the compound or nucleicacid of (a) either before, during and/or after the co-expressing thenucleic acid of the invention in the cell; and

(c) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell,

wherein an intact scaffold protein comprising the detectable moiety isexpressed (or is substantially expressed) on the extracellular surfaceof the cell when the cell's ER and trans-Golgi network is not inhibited.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ofthe invention in the cell and not adding the compound to be screened asan inhibitor to one of the divided cell samples.

In alternative embodiments the methods of the invention further compriserunning a positive control comprising dividing the plurality of thecells co-expressing the nucleic acid in the cell and adding a knowninhibitor of the cell's ER and/or trans-Golgi network to one of thedivided cell samples.

In alternative embodiments the transcriptional regulatory unit comprisesa promoter, an inducible promoter or a constitutive promoter.

In alternative embodiments the cell is a mammalian cell, a monkey cellor a human cell.

In alternative embodiments the scaffold proteins of the inventioncomprise all or part of a mouse Lyt2 or a human CD8 polypeptide.

In alternative embodiments the detectable moiety comprises an epitopefor an antibody, or a FLAG tag. The detectable moiety can be detected ormeasured on the extracellular surface of the cell by a high throughputscreen, a flow cytometry or microscope visualization.

In alternative embodiments the compound to be screened as an inhibitorof protease comprises a small molecule, a nucleic acid, a polypeptide orpeptide, a peptidomimetic, a polysaccharide or a lipid. In alternativeembodiments the compound to be screened as an inhibitor of protease is amember of a library of compounds to be screened, or a member of a randompeptide library or a chemical compound library.

In alternative embodiments the protease is an HIV-1 protease (PR). Theprotease can be a viral, a microbial or a mammalian protease.

The invention provides isolated, recombinant or synthetic nucleic acidsencoding a scaffold protein operatively linked to a transcriptionalregulatory unit, wherein the scaffold protein comprises:

(a) (i) an amino acid motif or subsequence susceptible to cleavage by aprotease under physiologic (cell culture) conditions;

(ii) a transmembrane domain;

(iii) a signal sequence or any amino acid motif that places the scaffoldprotein on the extracellular surface of the cell; and

(iv) a detectable moiety; or

(b) the nucleic acid of (a), wherein the scaffold protein furthercomprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,

wherein the ER retention motif or KDEL motif is positioned in thescaffold protein such that when PR is active the scaffold will beseparated into two pieces, leaving the ER retention motif-comprising orKDEL motif-comprising portion of the polypeptide in the ER and freeingthe detectable moiety-comprising portion to the cell's extracellularmembrane, and if PR is blocked or inactive, the entire scaffoldpolypeptide will be retained in the ER, and as a consequence will not bedetected on the cell's extracellular surface.

In alternative embodiments the protease is an HIV-1 protease (PR), orthe protease is a viral, a microbial or a mammalian protease.

In alternative embodiments the scaffold proteins of the inventioncomprise all or part of a mouse Lyt2 or a human CD8 polypeptide.

In alternative embodiments the detectable moiety comprises an epitopefor an antibody, or a FLAG tag.

The invention provides vectors, expression cassettes, cosmids orplasmids comprising the isolated, recombinant or synthetic nucleic acidsof the invention, and cells comprising any one, several or all of theseembodiments.

The invention provides isolated, recombinant or synthetic polypeptides,e.g., the so-called “scaffold proteins” of the invention, encoded by anucleic acid of the invention.

The invention provides cells comprising the isolated, recombinant orsynthetic nucleic acid of the invention, the vector, expressioncassette, cosmid or plasmid of the invention, or isolated, recombinantor synthetic polypeptide of the invention.

A chimeric (e.g., recombinant) polypeptide comprising:

-   -   (i) an amino acid motif or subsequence susceptible to cleavage        by the HIV-1 protease (PR) under physiologic (cell culture)        conditions;    -   (ii) a transmembrane domain;    -   (iii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iv) a detectable moiety,

wherein the amino acid motif or subsequence susceptible to cleavage bythe HIV-1 protease (PR) is positioned within the scaffold protein suchthat when the detectable moiety is cleavage away from (off from) thescaffold protein by the HIV-1 protease (PR) the remaining subsequence ofscaffold protein on the extracellular surface of the cell lacks thedetectable moiety;

(2) the chimeric polypeptide of (1), wherein the scaffold proteinfurther comprises an endoplasmic reticulum (ER) retention motif or aKDEL motif,

wherein the ER retention motif or KDEL motif is positioned in thescaffold protein such that when PR is active the scaffold will beseparated into two pieces, leaving the ER retention motif-comprising orKDEL motif-comprising portion of the polypeptide in the ER and freeingthe detectable moiety-comprising portion to the cell's extracellularmembrane, and if PR is blocked or inactive, the entire scaffoldpolypeptide will be retained in the ER, and as a consequence will not bedetected on the cell's extracellular surface; or

(3) the chimeric polypeptide of (1), wherein the scaffold proteinfurther comprises a p2/p7 recognition site imbedded in the cytoplasmicloop of the scaffold,

wherein optionally the p2/p7 recognition sequence comprises or consistsof ATIMMQRGN (SEQ ID NO:2), or optionally an exemplary amino-acidsequence of p2/p7 comprisesAEAMSQVTNS/ATIMMQRGN/FRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWK CGKEGHQMKDCTERQANATIMMQRGN (SEQ ID NO:3).

A chimeric (e.g., recombinant) polypeptide comprising:

-   -   (i) an amino acid motif or subsequence susceptible to cleavage        by the protease under physiologic (cell culture) conditions;    -   (ii) a transmembrane domain;    -   (iii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iv) a detectable moiety,

wherein the amino acid motif or subsequence susceptible to cleavage bythe protease is positioned within the scaffold protein such that whenthe detectable moiety is cleavage away from (off from) the scaffoldprotein by the protease the remaining subsequence of scaffold protein onthe extracellular surface of the cell lacks the detectable moiety; or

(2) the chimeric polypeptide of (1), wherein the scaffold proteinfurther comprises an endoplasmic reticulum (ER) retention motif or aKDEL (SEQ ID NO:1) motif,

wherein the ER retention motif or KDEL motif is positioned in thescaffold protein such that when PR is active the scaffold will beseparated into two pieces, leaving the ER retention motif-comprising orKDEL motif-comprising portion of the polypeptide in the ER and freeingthe detectable moiety-comprising portion to the cell's extracellularmembrane, and if PR is blocked or inactive, the entire scaffoldpolypeptide will be retained in the ER, and as a consequence will not bedetected on the cell's extracellular surface.

A chimeric (e.g., recombinant) polypeptide comprising:

-   -   (i) a transmembrane domain;    -   (ii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iii) a detectable moiety; or

(2) the chimeric polypeptide of (1), wherein the scaffold proteinfurther comprises an endoplasmic reticulum (ER) retention motif or aKDEL (SEQ ID NO:1) motif,

wherein the ER retention motif or KDEL motif is positioned in thescaffold protein such that when PR is active the scaffold will beseparated into two pieces, leaving the ER retention motif-comprising orKDEL motif-comprising portion of the polypeptide in the ER and freeingthe detectable moiety-comprising portion to the cell's extracellularmembrane, and if PR is blocked or inactive, the entire scaffoldpolypeptide will be retained in the ER, and as a consequence will not bedetected on the cell's extracellular surface.

In one embodiment, assays and cells of the invention can be used toidentify whether or not a viral protease (PR) is active. This is basedon the detection of a tag on the surface of a mammalian cell, e.g., ahuman cell. If PR is active, no tag will be detected. If PR is inactiveor blocked, the tag will appear on the surface of the cell. This can beeasily recognized with antibodies that are coupled to a fluorescent dyethrough a technique referred to as flow cytometry. As a result, analysisof various drugs on the expression of the tag on the surface of the cellcan be conducted. If the drug inhibits or blocks PR, the cells willbecome positive for tag surface expression. In contrast, if the drug isnot active against PR, the cells will be negative for tag expression.

In one embodiment, assays and cells of the invention rely on theengineering of a recombinant polypeptide, the so-called “scaffoldprotein”. In one embodiment, this scaffold is referred to as p2/7; whichis one of the natural targets of PR, and PR cleaves it at the boundarybetween p2 and p7 during normal HIV-1 infection. PR is essential for theviral lifecycle.

Drugs against PR can hinder its capacity to cleave its targets,resulting in inhibitory effects on viral replication. Thus, in someembodiments of this invention a p2/p7 scaffold is engineered in such away that instead of being expressed inside the cell, it is expressed onthe surface of the cell. To achieve surface expression, a “scaffoldprotein” such as the exemplary p2/p7 is fused to a signal (e.g., signalsequence) that directs it to the cell surface. Addition of anothersegment; a transmembrane domain (or motif), allows it to becomeincorporated into the cell surface. A marker (tag) is added to the p2/p7scaffold to allow detection of its presence on the surface.

In one embodiment, assays and cells of the invention are adapted to ahigh throughput system, where they can be used e.g., in the search ofmuch needed novel drugs against PR.

In alternative embodiments, methods of the invention are practiced invitro or in vivo. For example, the cells need not be intact for membraneanalysis of the presence or absence of detectable moieties.

In alternative embodiments, the invention provides cell-based methods(e.g., wherein the cell is a lymphocyte, such as a T cell, e.g., a CD4⁺cell) for monitoring the activity of an enzyme, e.g., a protease, e.g.,an HIV protease, e.g., an HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4Aprotease of hepatitis C virus, or HCV), comprising:

(1) (a) providing a nucleic acid encoding a scaffold protein operativelylinked to a transcriptional regulatory unit, wherein the scaffoldprotein comprises:

-   -   (i) an amino acid motif or subsequence susceptible to cleavage        by the enzyme, e.g., a protease, e.g., an HIV protease, e.g., an        HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A protease of        hepatitis C virus, under physiologic (e.g., cell culture)        conditions;    -   (ii) a transmembrane domain;    -   (iii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iv) a detectable moiety (e.g., a Green Fluorescent Protein        (GFP) or a luciferase, or any compound that can be directly or        indirectly detected),    -   wherein in alternative embodiments the amino acid motif or        subsequence susceptible to cleavage by the enzyme, e.g., the        protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an        NS2/NS3 or NS3/NS4A HCV protease, is positioned within the        scaffold protein such that when the detectable moiety (e.g.,        Green Fluorescent Protein (GFP) or luciferase) is cleaved away        from (off from) the scaffold protein by the enzyme, e.g.,        protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an        NS2/NS3 or NS3/NS4A HCV protease, and the remaining subsequence        of scaffold protein on the extracellular surface of the cell        lacks the detectable moiety;

(b) providing a nucleic acid encoding the enzyme, e.g., the protease,e.g., HIV protease, e.g., HIV-1 protease (PR), or NS2/NS3 or NS3/NS4AHCV protease, operatively linked to a transcriptional regulatory unit,or a cell that expresses a heterologous or endogenous enzyme, e.g.,protease, e.g., HIV protease, e.g., HIV-1 protease (PR);

(c) inserting (transfecting) the nucleic acid of step (b) (e.g.,comprising a nucleic acid encoding the enzyme, e.g., the protease, e.g.,HIV protease, e.g., HIV-1 protease) into the cell (e.g., a lymphocyte,such as a T cell, e.g., a CD4⁺ cell) if the cell does not alreadyexpress a heterologous or endogenous enzyme, e.g., protease, e.g., HIVprotease, e.g., HIV-1 protease (PR));

(d) co-expressing the nucleic acid of step (a) and (b) in the cell, orexpressing the nucleic acid of (a) in the cell if the cell alreadyexpresses a heterologous or endogenous HIV-1 protease (PR); and

(e) determining whether the scaffold protein comprising the detectablemoiety (e.g., a Green Fluorescent Protein (GFP) or a luciferase) isexpressed on the extracellular surface of the cell (e.g., by flowcytometry or any high-throughput assay),

wherein in alternative embodiments an intact scaffold protein comprisingthe detectable moiety is expressed on the extracellular surface of thecell when the enzyme, e.g., protease, e.g., HIV protease, e.g., HIV-1protease (PR), or NS2/NS3 or NS3/NS4A HCV protease, is not enzymaticallyactive, and an intact scaffold protein is not or is substantially lessexpressed on the extracellular surface of the cell when the enzyme,e.g., protease, e.g., HIV protease, e.g., HIV-1 protease (PR), orNS2/NS3 or NS3/NS4A HCV protease, is enzymatically active (thedetectable moiety is cleaved off by the enzyme, e.g., protease, e.g.,HIV protease, e.g., HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A HCVprotease);

(2) the method of (1), wherein the scaffold protein further comprises anendoplasmic reticulum (ER) retention motif or a KDEL motif,

wherein in alternative embodiments the ER retention motif or KDEL motifis positioned in the scaffold protein such that when enzyme, e.g.,protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or NS2/NS3 orNS3/NS4A HCV protease, is active the scaffold will be separated into twopieces, leaving the ER retention motif-comprising or KDELmotif-comprising portion of the polypeptide in the ER and freeing thedetectable moiety-comprising portion to the cell's extracellularmembrane, and if the enzyme, e.g., protease, e.g., HIV protease, e.g.,HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A HCV protease, is blocked orinactive, the entire scaffold polypeptide will be retained in the ER,and as a consequence will not be detected on the cell's extracellularsurface; or

(3) the method of (1), wherein the scaffold protein further comprises ap2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold;wherein optionally the p2/p7 recognition sequence comprises or consistsof ATIMMQRGN (SEQ ID NO:2), or optionally an exemplary amino-acidsequence of p2/p7 comprisesAEAMSQVTNS/ATIMMQRGN/FRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWK CGKEGHQMKDCTERQANATIMMQRGN (SEQ ID NO:3).

In alternative embodiments the methods of the invention further comprisescreening for an inhibitor of an enzyme, e.g., protease, e.g., HIVprotease, e.g., HIV-1 protease (PR) by:

(a) providing a compound to be screened as an inhibitor of an enzyme,e.g., protease, e.g., HIV protease, e.g., HIV-1 protease (PR), orNS2/NS3 or NS3/NS4A HCV protease, or providing a nucleic acid to bescreened as encoding an inhibitor of an enzyme, e.g., protease, e.g.,HIV protease, e.g., HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A HCVprotease;

(b) contacting a plurality of the cells (e.g., lymphocytes, such as Tcells, e.g., CD4+ cells) with the compound or nucleic acid of (a) eitherbefore, during and/or after the co-expressing the nucleic acid in thecell; and

(c) determining whether the scaffold protein comprising the detectablemoiety (e.g., a Green Fluorescent Protein (GFP) or a luciferase) isexpressed on the extracellular surface of the cell (e.g., by flowcytometry or any high-throughput assay),

wherein in alternative embodiments an intact scaffold protein comprisingthe detectable moiety is expressed on the extracellular surface of thecell when the enzyme, e.g., protease, e.g., HIV protease, e.g., HIV-1protease (PR), or NS2/NS3 or NS3/NS4A HCV protease, is inhibited by: thecompound, a composition encoded by the nucleic acid, or a compoundpresent in the cell only because the nucleic acid was expressed, and anintact scaffold protein is not or is substantially less expressed (inquantitative terms) on the extracellular surface of the cell when theenzyme, e.g., protease, e.g., HIV protease, e.g., HIV-1 protease (PR),or NS2/NS3 or NS3/NS4A HCV protease, is enzymatically active; forexample, in one embodiment the detectable moiety is cleaved off by theenzyme, e.g., protease, e.g., HIV protease, e.g., HIV-1 protease (PR),or NS2/NS3 or NS3/NS4A HCV protease, and the enzymatic activity of theenzyme, e.g., protease, e.g., HIV protease, e.g., HIV-1 protease (PR),or NS2/NS3 or NS3/NS4A HCV protease, is not significantly inhibited by:the compound, a composition encoded by the nucleic acid or a compoundpresent in the cell only because the nucleic acid was expressed.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ornucleic acids in the cell (e.g., as noted above, co-expressing thenucleic acid of step (a) and (b) in the cell, or expressing the nucleicacid of (a) in the cell if the cell already expresses a heterologous orendogenous enzyme, e.g., protease, e.g., HIV protease, e.g., HIV-1protease (PR), or NS2/NS3 or NS3/NS4A HCV protease, and not adding thecompound to be screened as an inhibitor to one of the divided cellsamples.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ornucleic acids in the cell (e.g., as noted above, co-expressing thenucleic acid of step (a) and (b) in the cell, or expressing the nucleicacid of (a) in the cell if the cell already expresses a heterologous orendogenous HIV-1 protease) and adding a known inhibitor of the enzyme,e.g., protease, e.g., HIV protease, e.g., HIV-1 protease (PR), orNS2/NS3 or NS3/NS4A HCV protease, to one of the divided cell samples.

In alternative embodiments, the amino acid motif or subsequencesusceptible to cleavage by the enzyme, e.g., protease, e.g., HIVprotease, e.g., HIV-1 protease (PR), under physiologic (cell culture)conditions comprises LEAQEEEEVGF (SEQ ID NO:3), or ATIMMQRGN (SEQ IDNO:2), or comprises a motif defined by and/or is definable by a protocolas described by Kurt (2003) Biophys J. 85(2):853-63; or Prabu-Jeyabalan(2002) Structure 10(3):369-81; or Prabu-Jeyabalan (2006) J. Virol.80(7):3607-16; or Kontijevskis (2007) Proteins 68(1):305-12; or Shoeman(1991) FEBS Lett. 278(2):199-203.

The HIV-1 protease (PR) can comprise or consist of SEQ ID NO:4 or SEQ IDNO:5, or an enzymatically active fragment thereof:

(SEQ ID NO: 4) PQVTLWQRPL VTIKIGGQLK EALLDTGADD TVLEEMSLPGRWKPKMIGGI GGFIKVRQYD QILIEICGHKAIGTVLVGPT PVNIIGRNLL TQIGCTLNF(SEQ ID NO: 5) PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF

The exemplary SEQ ID NO:5 is a prototype of an NL4-3 genome, but can beused with any other sequences from other strains of HIV.

In alternative embodiments the transcriptional regulatory unit comprisesa promoter, an inducible promoter or a constitutive promoter.

In alternative embodiments the cell is a mammalian cell, a monkey cellor a human cell, or the cell is a lymphocyte such as a T cell, e.g., aCD4′ or CD8′ cell.

In alternative embodiments the scaffold proteins of the inventioncomprise all or part of a mouse Lyt2 or a human CD8 polypeptide.

In alternative embodiments the detectable moiety comprises an epitopefor an antibody, or a FLAG tag. The detectable moiety can be detected ormeasured on the extracellular surface of the cell by a high throughputscreen, a flow cytometry or microscope visualization.

In alternative embodiments the compound to be screened as an inhibitorof enzyme, e.g., protease, e.g., HIV protease, e.g., HIV-1 protease(PR), or NS2/NS3 or NS3/NS4A HCV protease, comprises a small molecule, anucleic acid, a polypeptide or peptide, a peptidomimetic, apolysaccharide or a lipid.

In alternative embodiments the compound to be screened as an inhibitorof enzyme, e.g., protease, e.g., HIV protease, e.g., HIV-1 protease(PR), or NS2/NS3 or NS3/NS4A HCV protease, is a member of a library ofcompounds (e.g., a peptide library) to be screened, or a member of arandom peptide library or a chemical compound library.

The invention provides cell-based methods (e.g., wherein the cell is alymphocyte, such as a T cell, e.g., a CD4 cell) for monitoring theactivity of a protease comprising:

(1) (a) providing a nucleic acid encoding a scaffold protein operativelylinked to a transcriptional regulatory unit, wherein the scaffoldprotein comprises:

-   -   (i) an amino acid motif or subsequence susceptible to cleavage        by the protease under physiologic (cell culture) conditions;    -   (ii) a transmembrane domain;    -   (iii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iv) a detectable moiety (e.g., a Green Fluorescent Protein        (GFP) or a luciferase, or any compound that can be directly or        indirectly detected),    -   wherein in alternative embodiments the amino acid motif or        subsequence susceptible to cleavage by the protease is        positioned within the scaffold protein such that when the        detectable moiety is cleaved away from (off from) the scaffold        protein by the protease the remaining subsequence of scaffold        protein on the extracellular surface of the cell lacks the        detectable moiety;

(b) providing a nucleic acid encoding the protease operatively linked toa transcriptional regulatory unit, or a cell that expresses aheterologous or endogenous protease;

(c) inserting (transfecting) the nucleic acid of (b) into the cell ifthe cell does not already express a heterologous or endogenous protease;

(d) co-expressing the nucleic acid of (a) and (b) in the cell, orexpressing the nucleic acid of (a) in the cell if the cell alreadyexpresses a heterologous or endogenous protease; and

(e) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell (e.g., byflow cytometry or any high-throughput assay),

wherein in alternative embodiments an intact scaffold protein comprisingthe detectable moiety is expressed on the extracellular surface of thecell when the protease is not enzymatically active, and an intactscaffold protein is not or is substantially less expressed on theextracellular surface of the cell when the protease is enzymaticallyactive (the detectable moiety is cleaved off by the protease); or

(2) the method of (1), wherein the scaffold protein further comprises anendoplasmic reticulum (ER) retention motif or a KDEL motif,

wherein in alternative embodiments the ER retention motif or KDEL motifis positioned in the scaffold protein such that when PR is active thescaffold will be separated into two pieces, leaving the ER retentionmotif-comprising or KDEL motif-comprising portion of the polypeptide inthe ER and freeing the detectable moiety-comprising portion to thecell's extracellular membrane, and if PR is blocked or inactive, theentire scaffold polypeptide will be retained in the ER, and as aconsequence will not be detected on the cell's extracellular surface.

In alternative embodiments the methods of the invention further comprisescreening for an inhibitor of a protease by:

(a) providing a compound to be screened as an inhibitor of protease, orproviding a nucleic acid to be screened as encoding an inhibitor ofprotease;

(b) contacting a plurality of the cells with the compound or nucleicacid of either before, during and/or after the co-expressing the nucleicacid in the cell; and

(c) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell (e.g., byflow cytometry or any high-throughput assay),

wherein in alternative embodiments an intact scaffold protein comprisingthe detectable moiety is expressed on the extracellular surface of thecell when the protease is inhibited by: the compound, a compositionencoded by the nucleic acid, or a compound present in the cell onlybecause the nucleic acid was expressed, and an intact scaffold proteinis not or is substantially less expressed on the extracellular surfaceof the cell when the protease is enzymatically active (the detectablemoiety is cleaved off by the protease) and the enzymatic activity of theprotease is not significantly inhibited by: the compound, a compositionencoded by the nucleic acid, or a compound present in the cell onlybecause the nucleic acid was expressed.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ornucleic acids (e.g., as noted above, co-expressing the nucleic acid ofstep (a) and (b) in the cell, or expressing the nucleic acid of (a) inthe cell if the cell already expresses a heterologous or endogenousHIV-1 protease) and not adding the compound to be screened as aninhibitor to one of the divided cell samples.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ornucleic acids in the cell and adding a known inhibitor of the proteaseto one of the divided cell samples.

In alternative embodiments the transcriptional regulatory unit comprisesa promoter, or an inducible promoter, or a constitutive promoter.

In alternative embodiments the cell is a mammalian cell, a monkey cellor a human cell.

In alternative embodiments the scaffold proteins of the inventioncomprise all or part of a mouse Lyt2 or a human CD8 polypeptide.

In alternative embodiments the detectable moiety comprises an epitopefor an antibody, or a FLAG tag. The detectable moiety can be detected ormeasured on the extracellular surface of the cell by a high throughputscreen, a flow cytometry or microscope visualization.

In alternative embodiments the compound to be screened as an inhibitorof protease comprises a small molecule, a nucleic acid, a polypeptide orpeptide, a peptidomimetic, a polysaccharide or a lipid. The compound tobe screened can be an inhibitor of protease is a member of a library ofcompounds to be screened, or a member of a random peptide library or achemical compound library.

In alternative embodiments the protease is an HIV-1 protease (PR), orthe protease is a viral, a microbial or a mammalian protease.

The invention provides cell-based methods for monitoring the activity ofa cell's (e.g., a lymphocyte, such as a T cell, e.g., a CD4⁺ cell) ERand/or trans-Golgi network or a cell's ER and/or monitoring the activityof a cell's ER and/or trans-Golgi transport proteins, comprising:

(1) (a) providing a nucleic acid encoding a scaffold protein operativelylinked to a transcriptional regulatory unit, wherein the scaffoldprotein comprises:

-   -   (i) a transmembrane domain;    -   (ii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iii) a detectable moiety (e.g., a Green Fluorescent Protein        (GFP) or a luciferase, or any compound that can be directly or        indirectly detected);

(b) inserting (transfecting) the scaffold protein-encoding nucleic acidof (a) into the cell;

(d) expressing the nucleic acid of (a); and

(e) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell (e.g., byflow cytometry or any high-throughput assay),

wherein in alternative embodiments the scaffold protein is expressed onthe extracellular surface of the cell when the activity of the cell's ERand trans-Golgi network is functioning; or

(2) the method of (1), wherein the scaffold protein further comprises anendoplasmic reticulum (ER) retention motif or a KDEL motif,

wherein in alternative embodiments the ER retention motif or KDEL motifis positioned in the scaffold protein such that when PR is active thescaffold will be separated into two pieces, leaving the ER retentionmotif-comprising or KDEL motif-comprising portion of the polypeptide inthe ER and freeing the detectable moiety-comprising portion to thecell's extracellular membrane, and if PR is blocked or inactive, theentire scaffold polypeptide will be retained in the ER, and as aconsequence will not be detected on the cell's extracellular surface.

In alternative embodiments the methods of the invention further comprisescreening for an inhibitor of the cell's ER and trans-Golgi network by:

(a) providing a compound or nucleic acid to be screened as an inhibitorof the cell's ER and trans-Golgi network;

(b) contacting a plurality of the cells with the compound or nucleicacid of (a) either before, during and/or after the co-expressing thenucleic acid of the invention in the cell; and

(c) determining whether the scaffold protein comprising the detectablemoiety is expressed on the extracellular surface of the cell,

wherein an intact scaffold protein comprising the detectable moiety isexpressed (or is substantially expressed) on the extracellular surfaceof the cell when the cell's ER and trans-Golgi network is not inhibited.

In alternative embodiments the methods of the invention further comprisedividing the plurality of the cells co-expressing the nucleic acid ofthe invention in the cell and not adding the compound to be screened asan inhibitor to one of the divided cell samples.

In alternative embodiments the methods of the invention further compriserunning a positive control comprising dividing the plurality of thecells co-expressing the nucleic acid in the cell and adding a knowninhibitor of the cell's ER and/or trans-Golgi network to one of thedivided cell samples.

In alternative embodiments the transcriptional regulatory unit comprisesa promoter, an inducible promoter or a constitutive promoter.

In alternative embodiments the cell is a mammalian cell, a monkey cellor a human cell.

In alternative embodiments the scaffold proteins of the inventioncomprise all or part of a mouse Lyt2 or a human CD8 polypeptide.

In alternative embodiments the detectable moiety comprises an epitopefor an antibody, or a FLAG tag. The detectable moiety can be detected ormeasured on the extracellular surface of the cell by a high throughputscreen, a flow cytometry or microscope visualization.

In alternative embodiments the compound to be screened as an inhibitorof protease comprises a small molecule, a nucleic acid, a polypeptide orpeptide, a peptidomimetic, a polysaccharide or a lipid. In alternativeembodiments the compound to be screened as an inhibitor of protease is amember of a library of compounds to be screened, or a member of a randompeptide library or a chemical compound library.

In alternative embodiments the enzyme, e.g., protease, is an HIV enzyme,e.g., an HIV-1 protease (PR), or a hepatitis enzyme, e.g., an NS2/NS3 orNS3/NS4A HCV protease. The protease can be a viral, a microbial or amammalian enzyme, e.g., a protease. The viral enzyme, e.g., protease,can be any retroviral enzyme, lentiviral enzyme, HIV enzyme, hepatitisenzyme or any viral protease or other enzyme.

The invention provides isolated, recombinant or synthetic nucleic acidsencoding a scaffold or other structural protein operatively linked to atranscriptional regulatory unit, wherein the scaffold or otherstructural protein comprises:

(a) (i) an amino acid motif or subsequence susceptible to cleavage by anenzyme, e.g., a protease, under physiologic (cell culture) conditions;

(ii) a transmembrane domain;

(iii) a signal sequence or any amino acid motif that places the scaffoldprotein on the extracellular surface of the cell; and

(iv) a detectable moiety (e.g., a Green Fluorescent Protein (GFP) or aluciferase, or any compound that can be directly or indirectlydetected); or

(b) the nucleic acid of (a), wherein the scaffold or other structuralprotein further comprises an endoplasmic reticulum (ER) retention motifor a KDEL motif,

wherein in alternative embodiments the ER retention motif or KDEL motifis positioned in the scaffold or other structural protein such that whenenzyme, e.g., protease or PR, is active the scaffold will be separatedinto two pieces, leaving the ER retention motif-comprising or KDELmotif-comprising portion of the polypeptide in the ER and freeing thedetectable moiety-comprising portion to the cell's extracellularmembrane, and if the enzyme, e.g., protease or PR, is blocked orinactive, the entire scaffold polypeptide will be retained in the ER,and as a consequence will not be detected on the cell's extracellularsurface.

In alternative embodiments the enzyme is a protease, e.g., an HIV-1protease (PR), or the protease is a viral, a microbial or a mammalianprotease.

In alternative embodiments the scaffold proteins comprise all or part ofa mouse Lyt2 or a human CD8 polypeptide.

In alternative embodiments the detectable moiety comprises an epitopefor an antibody, or a FLAG tag.

The invention provides vectors, expression cassettes, cosmids orplasmids comprising the isolated, recombinant or synthetic nucleic acidsor the invention or used to practice this invention, and cellscomprising any one, several or all of these embodiments.

The invention provides isolated, recombinant or synthetic polypeptides,e.g., the so-called “scaffold proteins” of the invention, encoded by anucleic acid of the invention.

The invention provides cells comprising the isolated, recombinant orsynthetic nucleic acid of the invention, the vector, expressioncassette, cosmid or plasmid of the invention, or isolated, recombinantor synthetic polypeptide of the invention.

A chimeric (e.g., recombinant) polypeptide comprising:

-   -   (i) an amino acid motif or subsequence susceptible to cleavage        by an enzyme, e.g., a protease or an HIV-1 protease (PR), or an        NS2/NS3 or NS3/NS4A protease of HCV, under physiologic (cell        culture) conditions;    -   (ii) a transmembrane domain;    -   (iii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iv) a detectable moiety (e.g., a Green Fluorescent Protein        (GFP) or a luciferase, or any compound that can be directly or        indirectly detected),

wherein in alternative embodiments the amino acid motif or subsequencesusceptible to cleavage by the enzyme, e.g., protease or HIV-1 protease(PR), or an NS2/NS3 or NS3/NS4A protease of HCV, is positioned withinthe scaffold protein such that when the detectable moiety is cleavedaway from (off from) the scaffold protein by the enzyme, e.g., proteaseor HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A protease of HCV, theremaining subsequence of scaffold protein on the extracellular surfaceof the cell lacks the detectable moiety;

(2) the chimeric polypeptide of (1), wherein the scaffold proteinfurther comprises an endoplasmic reticulum (ER) retention motif or aKDEL motif,

wherein in alternative embodiments the ER retention motif or KDEL motifis positioned in the scaffold protein such that when the enzyme, e.g.,protease or HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A protease ofHCV, is active the scaffold will be separated into two pieces, leavingthe ER retention motif-comprising or KDEL motif-comprising portion ofthe polypeptide in the ER and freeing the detectable moiety-comprisingportion to the cell's extracellular membrane, and if the enzyme, e.g.,protease or HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A protease ofHCV, is blocked or inactive, the entire scaffold polypeptide will beretained in the ER, and as a consequence will not be detected on thecell's extracellular surface; or

(3) the chimeric polypeptide of (1), wherein the scaffold proteinfurther comprises a p2/p7 recognition site imbedded in the cytoplasmicloop of the scaffold,

wherein in alternative embodiments the p2/p7 recognition sequencecomprises or consists of ATIMMQRGN (SEQ ID NO:2), or optionally anexemplary amino-acid sequence of p2/p7 comprisesAEAMSQVTNS/ATIMMQRGN/FRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWK CGKEGHQMKDCTERQANATIMMQRGN (SEQ ID NO:3).

A chimeric (e.g., recombinant) polypeptide comprising:

-   -   (i) an amino acid motif or subsequence susceptible to cleavage        by an enzyme, e.g., protease or HIV-1 protease (PR), or an        NS2/NS3 or NS3/NS4A protease of HCV, under physiologic (cell        culture) conditions;    -   (ii) a transmembrane domain;    -   (iii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iv) a detectable moiety (e.g., a Green Fluorescent Protein        (GFP) or a luciferase, or any compound that can be directly or        indirectly detected),

wherein in alternative embodiments the amino acid motif or subsequencesusceptible to cleavage by the enzyme, e.g., protease or HIV-1 protease(PR), or NS2/NS3 or NS3/NS4A protease of HCV, is positioned within thescaffold protein such that when the detectable moiety is cleaved awayfrom (off from) the scaffold protein by the enzyme, e.g., protease orHIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, theremaining subsequence of scaffold protein on the extracellular surfaceof the cell lacks the detectable moiety; or

(2) the chimeric polypeptide of (1), wherein the scaffold proteinfurther comprises an endoplasmic reticulum (ER) retention motif or aKDEL motif,

wherein in alternative embodiments the ER retention motif or KDEL motifis positioned in the scaffold protein such that when the enzyme, e.g.,protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV,is active the scaffold will be separated into two pieces, leaving the ERretention motif-comprising or KDEL motif-comprising portion of thepolypeptide in the ER and freeing the detectable moiety-comprisingportion to the cell's extracellular membrane, and if the enzyme, e.g.,protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV,is blocked or inactive, the entire scaffold polypeptide will be retainedin the ER, and as a consequence will not be detected on the cell'sextracellular surface.

A chimeric (e.g., recombinant) polypeptide comprising:

-   -   (i) a transmembrane domain;    -   (ii) a signal sequence or any amino acid motif that places the        scaffold protein on the extracellular surface of the cell; and    -   (iii) a detectable moiety (e.g., a Green Fluorescent Protein        (GFP) or a luciferase, or any compound that can be directly or        indirectly detected); or

(2) the chimeric polypeptide of (1), wherein the scaffold proteinfurther comprises an endoplasmic reticulum (ER) retention motif or aKDEL motif,

wherein in alternative embodiments the ER retention motif or KDEL motifis positioned in the scaffold protein such that when PR is active thescaffold will be separated into two pieces, leaving the ER retentionmotif-comprising or KDEL motif-comprising portion of the polypeptide inthe ER and freeing the detectable moiety-comprising portion to thecell's extracellular membrane, and if PR is blocked or inactive, theentire scaffold polypeptide will be retained in the ER, and as aconsequence will not be detected on the cell's extracellular surface.

In one embodiment, assays and cells of the invention can be used toidentify whether or not an enzyme, e.g., protease or HIV-1 protease(PR), or NS2/NS3 or NS3/NS4A protease of HCV, is active. This is basedon the detection of a tag on the surface of a mammalian cell, e.g., ahuman cell. If the enzyme, e.g., protease or HIV-1 protease (PR), orNS2/NS3 or NS3/NS4A protease of HCV, is active, no tag will be detected.If the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 orNS3/NS4A protease of HCV, is inactive or blocked, the tag will appear onthe surface of the cell. This can be easily recognized with antibodiesthat are coupled to a fluorescent dye through a technique referred to asflow cytometry. As a result, analysis of various drugs on the expressionof the tag on the surface of the cell can be conducted. If the druginhibits or blocks the enzyme, e.g., protease or HIV-1 protease (PR), orNS2/NS3 or NS3/NS4A protease of HCV, the cells will become positive fortag surface expression. In contrast, if the drug is not active againstthe enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 orNS3/NS4A protease of HCV, the cells will be negative for tag expression.

In one embodiment, assays and cells of the invention rely on theengineering of a recombinant polypeptide, the so-called “scaffoldprotein”. In one embodiment, this scaffold is referred to as p2/7; whichis one of the natural targets of PR, and PR cleaves it at the boundarybetween p2 and p7 during normal HIV-1 infection. PR is essential for theviral lifecycle.

Drugs against the enzyme, e.g., protease or HIV-1 protease (PR), orNS2/NS3 or NS3/NS4A protease of HCV, can hinder its capacity to cleaveits targets, resulting in inhibitory effects on viral replication. Thus,in some embodiments of this invention a p2/p7 scaffold is engineered insuch a way that instead of being expressed inside the cell, it isexpressed on the surface of the cell. To achieve surface expression, a“scaffold protein” such as the exemplary p2/p7 is fused to a signal(e.g., signal sequence) that directs it to the cell surface. Addition ofanother segment; a transmembrane domain (or motif), allows it to becomeincorporated into the cell surface. A marker (tag) is added to the p2/p7scaffold to allow detection of its presence on the surface.

In one embodiment, assays and cells of the invention are adapted to ahigh throughput system, where they can be used e.g., in the search ofmuch needed novel drugs against the enzyme, e.g., protease or HIV-1protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV.

In alternative embodiments, methods of the invention are practiced invitro or in vivo. For example, the cells need not be intact for membraneanalysis of the presence or absence of detectable moieties (e.g., GreenFluorescent Protein (GFP), luciferase or any compound that can bedirectly or indirectly detected).

Cell-Based Methods and Multiplexed Systems

In alternative embodiments, the invention provides cell-based methodsand multiplexed systems for monitoring the activity of any enzyme, e.g.,protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV,comprising:

(1) (a) providing: a nucleic acid encoding a chimeric (hybrid) proteinoperatively linked to a transcriptional regulatory unit (e.g., apromoter); and, a cell comprising an environment capable of supportingthe expression of the chimeric (hybrid) protein by the nucleic acid,

wherein the chimeric (hybrid) protein comprises a chimeric Gal4expression system comprising (i) an N-terminal Gal4 DNA-binding domain(e.g., DBD: aa 1-147); (ii) an enzyme whose activity is to be monitored,or an enzymatically active fragment thereof; and (iii) a Gal4 C-terminalTransactivation domain (e.g., TAD: aa 768-881),

and the enzyme whose activity is to be monitored or the enzymaticallyactive fragment thereof is positioned in or within the chimeric proteinsuch that an enzymatically active enzyme or enzymatically activefragment thereof is capable of cleaving or physically separating orotherwise functionally separating the N-terminal Gal4 DNA-binding domainfrom the Gal4 C-terminal Transactivation domain such that the Gal4 canno longer act as a functional transcription factor, and if the enzymewhose activity is to be monitored is inhibited such that it is no longerenzymatically active (or substantially no longer enzymatically active)the Gal4 C-terminal Transactivation domain in conjunction with theN-terminal Gal4 DNA-binding domain can function as a functionaltranscription factor;

(b) inserting (transfecting) the nucleic acid of (a) into the cell,

wherein optionally the cell does not already express a heterologous orendogenous enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 orNS3/NS4A protease of HCV; and

(c) contacting the cell with a putative (test) enzyme inhibitor,

wherein optionally the enzyme inhibitor comprises a small molecule, aprotein, a nucleic acid, a polysaccharide and/or a lipid,

and optionally the enzyme inhibitor is added to the cell before, duringand/or after inserting (transfecting) the nucleic acid of (a) into thecell and/or expressing the chimeric protein encoded by the nucleic acidof (a) in the cell,

and optionally cell-based method further comprises a negative controlset of cells into which the nucleic acid of (a) also has been insertedand transfected and expresses the chimeric protein encoded by thenucleic acid of (a), but the negative control set of cells is notexposed to the putative (test) enzyme inhibitor or is exposed to adifferent putative (test) enzyme inhibitor;

(d) determining whether the putative (test) enzyme inhibitor is aneffective or sufficient inhibitor of the enzyme or enzymatically activefragment thereof by measuring the ability of the Gal4 C-terminalTransactivation domain in conjunction with the N-terminal Gal4DNA-binding domain to function as a functional transcription factor;

(2) the method of (1), wherein the enzyme is a protease or an HIV-1protease (PR), or a NS2/NS3 or NS3/NS4A protease of HCV; or

(3) the method of (1), wherein the cell is a lymphocyte or a T cell, ora CD4+ T cell, or a human cell.

In alternative embodiments the methods further comprise running anegative control comprising dividing the plurality of the cellsco-expressing the nucleic acid of (a) in the cell and not adding thecompound to be screened (the putative (test) enzyme inhibitor) as aninhibitor to one of the divided cell samples.

In alternative embodiments the methods further comprise running apositive control comprising dividing the plurality of the cellsco-expressing the nucleic acid of (a) in the cell and adding a knowninhibitor of the enzyme, e.g., a known inhibitor of a protease or anHIV-1 protease (PR), or a NS2/NS3 or NS3/NS4A protease of HCV, to one ofthe divided cell samples.

In alternative embodiments of the methods, the transcriptionalregulatory unit comprises a promoter, an inducible promoter or aconstitutive promoter.

In alternative embodiments of the methods, the cell is a mammalian cell,a monkey cell or a human cell.

In alternative embodiments of the methods, the positive activity of theGal4 C-terminal Transactivation domain in conjunction with theN-terminal Gal4 DNA-binding domain to function as a functionaltranscription factor is detected or measured by a high throughputscreen, a flow cytometry or microscope visualization.

In alternative embodiments of the methods, the (“test”) compound to bescreened as an inhibitor of the enzyme, e.g., protease or HIV-1 protease(PR), or NS2/NS3 or NS3/NS4A protease of HCV, comprises a smallmolecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, apolysaccharide or a lipid.

In alternative embodiments of the methods, the compound to be screenedas an inhibitor of the enzyme, e.g., protease or HIV-1 protease (PR), orNS2/NS3 or NS3/NS4A protease of HCV, is a member of a library ofcompounds to be screened, or a member of a random peptide library or achemical compound library.

In alternative embodiments of the methods, the transcriptionalregulatory unit comprises a promoter, e.g., an inducible promoter or aconstitutive promoter.

In alternative embodiments of the methods, the cell is a mammalian cell,a monkey cell or a human cell; or a lymphocyte, or a T cell, or a CD4-or CD8-expressing cell.

In alternative embodiments of the methods, the enzyme is a viralprotease, a microbial protease or a mammalian protease.

In alternative embodiments the invention provides isolated, recombinantor synthetic nucleic acids encoding a chimeric (hybrid) proteinoperatively linked to a transcriptional regulatory unit (e.g., apromoter)

wherein the chimeric (hybrid) protein comprises a chimeric Gal4expression system comprising (i) an N-terminal Gal4 DNA-binding domain(e.g., DBD: aa 1-147); (ii) an enzyme whose activity is to be monitored,or an enzymatically active fragment thereof; and (iii) a Gal4 C-terminalTransactivation domain (e.g., TAD: aa 768-881),

and the enzyme whose activity is to be monitored or the enzymaticallyactive fragment thereof is positioned in or within the chimeric proteinsuch that an enzymatically active enzyme or enzymatically activefragment thereof is capable of cleaving or physically separating orotherwise functionally separating the N-terminal Gal4 DNA-binding domainfrom the Gal4 C-terminal Transactivation domain such that the Gal4 canno longer act as a functional transcription factor, and if the enzymewhose activity is to be monitored is inhibited such that it is no longerenzymatically active (or substantially no longer enzymatically active)the Gal4 C-terminal Transactivation domain in conjunction with theN-terminal Gal4 DNA-binding domain can function as a functionaltranscription factor.

In alternative embodiments the invention provides cells comprising theisolated, recombinant or synthetic nucleic acid of the invention.

In alternative embodiments the invention provides vectors, expressioncassettes, cosmids or plasmids comprising or having contained thereinthe isolated, recombinant or synthetic nucleic acid of the invention.

In alternative embodiments the invention provides chimeric Gal4expression systems comprising a polypeptide comprising (i) an N-terminalGal4 DNA-binding domain (e.g., DBD: aa 1-147); (ii) an enzyme whoseactivity is to be monitored, or an enzymatically active fragmentthereof; and (iii) a Gal4 C-terminal Transactivation domain (e.g., TAD:aa 768-881), and the enzyme whose activity is to be monitored or theenzymatically active fragment thereof is positioned in or within thechimeric protein such that an enzymatically active enzyme orenzymatically active fragment thereof is capable of cleaving orphysically separating or otherwise functionally separating theN-terminal Gal4 DNA-binding domain from the Gal4 C-terminalTransactivation domain such that the Gal4 can no longer act as afunctional transcription factor, and if the enzyme whose activity is tobe monitored is inhibited such that it is no longer enzymatically active(or substantially no longer enzymatically active) the Gal4 C-terminalTransactivation domain in conjunction with the N-terminal Gal4DNA-binding domain can function as a functional transcription factor.

In alternative embodiments the enzyme is a viral protease, a microbialprotease or a mammalian protease, or the enzyme is an HIV-1 protease(PR), or a NS2/NS3 or a NS3/NS4A protease.

In alternative aspects, compositions and methods of the invention arepracticed in vitro or in vivo. For example, the cells need not be intactfor analysis of the effectiveness of a test compound to be sufficientlyeffective in inhibiting the activity of an enzyme, e.g., an HIVprotease.

In alternative aspects, compositions and methods of the invention arepracticed as multiplexed systems adapted for multiplexed analysis of aplurality of enzyme (more than one enzyme) inhibitors or modulators,comprising: a chimeric Gal4 expression system of the invention, or acell of the invention, or the cell-based method for monitoring theactivity of any enzyme of the invention, wherein inhibition of differentenzymes is monitored by the expression of a different detectable moiety,e.g., a different Fluorescent Protein (FP), e.g., an e-green fluorescentprotein, or eGFP (excited with the 488 nm blue laser, an e-cyanfluorescent protein (or eCFP, using a 405 nm violet laser), and/or anmOrange or an mCherry (561 nm yellow laser).

Cell-Based Assays for the Identification of Compositions that InhibitEnvelop Processing

In alternative embodiments, the invention provides cell-based assays toscreen for compositions, e.g., small molecules or drugs, that inhibit ormodify the activity of enzymes such as calcium-dependent proteinconvertases involved in HIV envelop protein processing, includingcleavage of the HIV gp160 envelope precursor, resulting in gp120 andgp41 envelope products.

In alternative embodiments, the invention provides isolated, recombinantor synthetic nucleic acids encoding a chimeric (hybrid) protein, whereinthe chimeric

(hybrid) protein comprises (or consists of) from N- to C-terminus:

(a) (i) a signal sequence (motif) for Endoplasmic Reticulum (ER)targeting,

(ii) a tag or detection moiety, or “scaffold”, capable of beingrecognized on a cell surface,

(iii) at least two transmembrane domains that span the ER membrane, withan extra loop at the ER luminal face,

(iv) an enzyme recognition/cleavage site spanning a segment of agp120/41 boundary, facing the ER lumen, and

(v) an ER retention sequence or motif;

(b) the chimeric (hybrid) protein of (a), wherein the tag or detectionmoiety, or “scaffold”, comprises a tag for an antibody or an antigenbinding fragment thereof (the antibody binding specifically to the tagor detection moiety, or “scaffold”), or the tag or detection moiety, or“scaffold”, comprises a ligand, or the tag or detection moiety, or“scaffold”, comprises a FLAG molecule or equivalent thereof;

(c) the chimeric (hybrid) protein of (a), wherein enzymerecognition/cleavage site comprises a furin enzyme recognition/cleavagesite, a calcium-dependent protein convertase enzyme recognition/cleavagesite, prohormone convertase-1 (PC1) enzyme recognition/cleavage site, oran enzyme recognition/cleavage site derived from a member of thesubtilisin/kexin family of proprotein convertases;

(d) the chimeric (hybrid) protein of (a), wherein enzymerecognition/cleavage site comprises an enzyme recognition/cleavage sitewithin the V3 loop of gp120;

(e) the chimeric (hybrid) protein of (a), wherein the ER retentionsequence or motif comprises a KDEL (SEQ ID NO:1) sequence or equivalentthereof;

(f) the chimeric (hybrid) protein of (a), wherein the gp120/41 is anHIV-1 gp120/41;

(g) the chimeric (hybrid) protein of (a), wherein the at least twotransmembrane domains that span the ER membrane consist of twotransmembrane domains;

(h) the chimeric (hybrid) protein of (a), wherein the at least twotransmembrane (TM) domains that span the ER membrane comprise at leastone TM of a CRR5;

(i) the chimeric (hybrid) protein of (h), wherein the at least twotransmembrane (TM) domains that span the ER membrane comprise TM1 andTM2 from the CRR5;

(j) the chimeric (hybrid) protein of (a), wherein the gp120/gp41boundary comprises the recognition/cleavage site REKRA (SEQ ID NO:14),

(k) the chimeric (hybrid) protein of (a), wherein the gp120/gp41boundary further comprises a restriction enzyme recognition site acidsat both sides, or comprises additional amino-acids at both sidescomprising AKRRVVQREKR (SEQ ID NO:15) and AVGIGALF (SEQ ID NO:16); or

(l) the isolated, recombinant or synthetic nucleic acid encoding thechimeric (hybrid) protein is operatively linked to a transcriptionalregulatory unit, or a promoter such as an inducible or constitutivepromoter.

In alternative embodiments, the invention provides vectors, recombinantviruses, cloning vehicles, expression cassettes, cosmids or plasmidscomprising (or consisting of) or having contained therein the isolated,recombinant or synthetic nucleic acid of the invention.

In alternative embodiments, the invention provides chimeric or hybridpolypeptides comprising (or consisting of): (a) the polypeptide encodedby the nucleic acid of the invention; or (b) the chimeric (hybrid)protein of (a), wherein the protein comprises a synthetic protein orpeptide, recombinant protein or peptide, a peptidomimetic or acombination thereof.

In alternative embodiments, the invention provides chimeric or hybridproteins comprising (or consisting of) from N- to C-terminus:

(a)) (i) a signal sequence (motif) for Endoplasmic Reticulum (ER)targeting,

(ii) a tag or detection moiety, or “scaffold”, capable of beingrecognized on a cell surface,

(iii) at least two transmembrane domains that span the ER membrane, withan extra loop at the ER luminal face,

(iv) an enzyme recognition/cleavage site spanning a segment of agp120/41 boundary, facing the ER lumen, and

(v) an ER retention sequence or motif;

(b) the chimeric (hybrid) protein of (a), wherein the tag or detectionmoiety, or “scaffold”, comprises a tag for an antibody or an antigenbinding fragment thereof (the antibody binding specifically to the tagor detection moiety, or “scaffold”), or the tag or detection moiety, or“scaffold”, comprises a ligand, or the tag or detection moiety comprisesa FLAG molecule or equivalent thereof;

(c) the chimeric (hybrid) protein of (a), wherein enzymerecognition/cleavage site comprises a furin enzyme recognition/cleavagesite, a calcium-dependent protein convertase enzyme recognition/cleavagesite, prohormone convertase-1 (PC1) enzyme recognition/cleavage site, oran enzyme recognition/cleavage site derived from a member of thesubtilisin/kexin family of proprotein convertases;

(d) the chimeric (hybrid) protein of (a), wherein enzymerecognition/cleavage site comprises an enzyme recognition/cleavage sitewithin the V3 loop of gp120;

(e) the chimeric (hybrid) protein of (a), wherein the ER retentionsequence or motif comprises a KDEL (SEQ ID NO:1) sequence or equivalentthereof;

(f) the chimeric (hybrid) protein of (a), wherein the gp120/41 is anHIV-1 gp120/41;

(g) the chimeric (hybrid) protein of (a), wherein the at least twotransmembrane domains that span the ER membrane consist of twotransmembrane domains;

(h) the chimeric (hybrid) protein of (a), wherein the at least twotransmembrane (TM) domains that span the ER membrane comprise at leastone TM of a CRR5;

(i) the chimeric (hybrid) protein of (h), wherein the at least twotransmembrane (TM) domains that span the ER membrane comprise TM1 andTM2 from the CRR5;

(j) the chimeric (hybrid) protein of (a), wherein the gp120/gp41boundary comprises the recognition/cleavage site REKRA (SEQ ID NO:2);

(k) the chimeric (hybrid) protein of (a), wherein the gp120/gp41boundary further comprises a restriction enzyme recognition site acidsat both sides, or comprises additional amino-acids at both sidescomprising AKRRVVQREKR (SEQ ID NO:15) and AVGIGALF (SEQ ID NO:16); or

(l) the chimeric (hybrid) protein of (a), wherein the protein comprises(or consists of) a synthetic protein or peptide, recombinant protein orpeptide, a peptidomimetic or a combination thereof.

In alternative embodiments, the invention provides cells comprising (a)an isolated, recombinant or synthetic nucleic acid of the invention; (b)a vector, recombinant virus, cloning vehicle, expression cassette,cosmid or plasmid of the invention; (c) a chimeric or hybrid polypeptideof the invention; or, (d) the cell of (a), (b) or (c), wherein the cellis a mammalian or a human cell.

In alternative embodiments, the invention provides cell-based methodsfor monitoring the activity of an enzyme, or for screening for aninhibitor of the enzyme, comprising:

(1) (a) providing: (i) a nucleic acid encoding the chimeric (hybrid)protein of the invention, or the nucleic acid of the invention,operatively linked to a transcriptional regulatory unit (e.g., apromoter, such as an inducible or constitutive promoter), or (ii) thevector, recombinant virus, cloning vehicle, expression cassette, cosmidor plasmid of the invention; and, a cell comprising an environmentcapable of supporting the expression of the chimeric (hybrid) protein bythe nucleic acid;

(b) inserting (e.g., transfecting or infecting) the nucleic acid,vector, recombinant virus, cloning vehicle, expression cassette, cosmidor plasmid of (a) into the cell; and

(c) contacting the cell with a putative (test) enzyme inhibitor,

wherein optionally the enzyme inhibitor is added to the cell before,during and/or after inserting (transfecting) the nucleic acid, vector,recombinant virus, cloning vehicle, expression cassette, cosmid orplasmid of (a) into the cell and/or expressing the chimeric proteinencoded by a nucleic acid of (a) in the cell,

and optionally the cell-based method further comprises a negative,positive and/or alternative control set of cells into which the nucleicacid, vector, recombinant virus, cloning vehicle, expression cassette,cosmid or plasmid of (a) also has been inserted (or transfected) andexpresses the chimeric protein encoded by a nucleic acid of (a), but thenegative control set of cells is not exposed to the putative (test)enzyme inhibitor or is exposed to a different putative (test) enzymeinhibitor, or a different amount of putative (test) enzyme inhibitor, ora positive control wherein the cells are exposed to a known inhibitor ofthe enzyme; and

(d) determining whether the putative (test) enzyme inhibitor is aneffective or sufficient inhibitor or modulator of the enzyme or anenzymatically active fragment thereof by measuring the ability of theputative (test) enzyme inhibitor to partially or completely inhibitcleavage of the enzyme recognition/cleavage site;

(2) measuring the ability of the putative (test) enzyme inhibitor topartially or completely inhibit cleavage of the enzymerecognition/cleavage site comprises detecting and/or measuring theamount of tag or detection moiety, or “scaffold”, on the cell surface;or

(3) the method of (1) or (2), wherein the cell is a lymphocyte or a Tcell, or a CD4+ T cell, or a human cell.

In one embodiment the method further comprises running a negativecontrol comprising dividing the plurality of the cells co-expressing anucleic acid of (a) in the cell and not adding the compound to bescreened (the putative (test) enzyme inhibitor) as an inhibitor to oneof the divided cell samples.

In one embodiment the method further comprises running a positivecontrol comprising dividing the plurality of the cells co-expressing thenucleic acid of (a) in the cell and adding a known inhibitor of theenzyme, e.g., a known inhibitor of a furin enzyme, a calcium-dependentprotein convertase enzyme, prohormone convertase-1 (PC1) enzyme, or anenzyme from a member of the subtilisin/kexin family of proproteinconvertases, to one of the divided cell samples.

In one embodiment of a method of the invention the transcriptionalregulatory unit comprises a promoter, an inducible promoter or aconstitutive promoter. The cell can be a mammalian cell, a monkey cellor a human cell.

In one embodiment of a method of the invention the tag or detectionmoiety, or “scaffold”, is detected or measured on the cell surface by ahigh throughput screen, a flow cytometry or microscope visualization.

In one embodiment of a method of the invention the compound to bescreened as an inhibitor of the enzyme comprises a small molecule, anucleic acid, a polypeptide or peptide, a peptidomimetic, apolysaccharide and/or a lipid. In one embodiment of a method of theinvention the compound to be screened as an inhibitor of the enzyme is amember of a library of compounds to be screened, or a member of a randompeptide library or a chemical compound library.

In one embodiment of a method of the invention the cell is a mammaliancell, a monkey cell or a human cell; or a lymphocyte, or a T cell, or aCD4- or CD8-expressing cell. In alternative aspects, methods of theinvention are practiced in vitro or in vivo. For example, the cells neednot be intact for analysis of the effectiveness of a test compound to besufficiently effective in inhibiting the activity of an enzyme.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 graphically illustrates a representation of an exemplary assay ofthe invention, as discussed in detail, below.

FIG. 2A graphically illustrates an exemplary vector used to corroboratethe expression of a scaffold of this invention on a cell surface by flowcytometry, as graphically illustrated in FIG. 2B, as discussed indetail, below.

FIG. 3 illustrates by fluorescence microscopy use of an exemplary vectorof the invention, a Tet-inducible HIV-based self-inactivating vector,that allows regulated expression of a gene of interest, as discussed indetail, below.

FIG. 4, lower panels, illustrate by fluorescence microscopy that aN-terminal GFP fusion is highly expressed, as illustrated in FIG. 4;and, this fusion seems to be expressed at low levels even in the absenceof inhibitor, as illustrated in FIG. 4, upper panels and FIG. 5,demonstrating that active PR can be expressed at low levels withoutcytotoxic effects, as discussed in detail, below.

FIG. 5 graphically illustrates flow cytometry analysis of cellsexpressing PR-GFP fusion, as discussed in detail, below.

FIG. 6 illustrates a Western blot analysis of protease (PR) activityusing the exemplary vector of FIG. 3, as discussed in detail, below.

FIG. 7 illustrates an exemplary construct of the invention and anexemplary scenario for monitoring of protease, e.g., PR, activity, asdiscussed in detail, below: briefly, when protease, e.g., PR, is active(left panel of FIG. 7), the scaffold will be separated into two pieces,leaving the KDEL portion in the ER and freeing the FLAG tag portion tothe membrane, where it will be detected by flow cytometry; if protease,e.g., PR, is blocked or inactive (right panel of FIG. 7), the entirescaffold will be retained in the ER, and as a consequence will not bedetected on the surface.

FIG. 8 illustrates HIV-1 Genome organization, as discussed in detail inExample 1, below.

FIG. 9 illustrates a fluorescence analysis showing indirect confirmationof HIV-1 PR activity: FIG. 9A illustrates exemplary plasmids pCMVD8.2,D8.7 and D8.91; and FIG. 9B illustrates a Western blot detecting thepresence of p24; as discussed in detail in Example 1, below

FIG. 10 illustrates an exemplary construct of the invention (LTR/MinCMV-TO/GFP/pPGK/rtTA/IRES/Puro/LTR) and the results of an inducibleexpression system of the invention, as discussed in detail in Example 1,below.

FIG. 11 and FIG. 12 graphically illustrate expression of exemplary HIV-1PR/GFP fusion proteins of the invention: in order to detect PRexpression, GFP was fused to the carboxy- (C-) or amino- (N-) terminusof PR, as discussed in detail in Example 1, below.

FIG. 12 illustrates a Western Blot of PR/GFP fusion proteins withanti-PR antibody, as discussed in detail in Example 1, below.

FIG. 13 illustrates targeting HIV-1 PR to different cellularcompartments: PR was targeted to the nucleus by adding a nuclearlocalization signal to the C terminus using the exemplary vector, asdiscussed in detail in Example 1, below.

FIG. 14 illustrates a schematic representation of an exemplary scaffoldof the invention and an exemplary protease (e.g., PR) assay based onsurface expression of the scaffold protein, as discussed in detail inExample 1, below.

FIG. 15 illustrates two exemplary constructs of the invention:pCMV/GFP=>Zeo, and pCMV/GFP=>SS-FLAG/P2-P7/TM=>Zeo; and flow cytometryanalysis of the expression of these two scaffold proteins on the surfaceof 293T cells; FIG. 15A illustrates results for the control constructand FIG. 15B illustrates results for the scaffold protein construct; asdiscussed in detail in Example 1, below.

FIG. 16 illustrates the results of use of an exemplary construct of theinvention, pCMV/GFP/PCS/GFP-NLS=Zeo, where GFP as a biosensor of PRactivity; as discussed in detail in Example 1, below.

FIG. 17 illustrates an exemplary construct of the invention where anuclear localization signal is separated by a detectable moiety, e.g., afluorescent protein, by a protease cleavage site; as discussed in detailin Example 1, below.

FIG. 18 schematically illustrates an exemplary assay of the inventionfor screening peptide libraries; as discussed in detail in Example 1,below.

FIG. 19 schematically illustrates an overview of an exemplary assay ofthe invention: FIG. 19A—Wild type Gal4 as control, no Dox; FIG. 19B—Inthe presence of Dox, Gal4 expression is induced; FIG. 19C—The samesystem with the PR/Gal4 fusion; FIG. 19D—The same scenario as in 19C butin the presence of a PR inhibitor; as discussed in detail in Example 2,below.

FIG. 20 illustrates a transient expression of components of an exemplaryassay in HEK293T cells: FIG. 20A schematically illustrates constructsused for transient expression of the assay elements; FIG. 20B: TopPanel: illustration of a fluorescence microscopy of eGFP expression inHEK293T cells 24 hours post transfection with the reporter vector (pFR),or co-transfection with the reporter vector plus either the Gal4,PR/Gal4, or PRm/Gal4 vectors; Bottom panel: HEK293T cells were analyzedby flow cytometry at 24 hours post transfection with the same conditionstop panel; FIG. 20C graphically illustrates the quantification of eGFPexpression in HEK293T transfected with various assay elements; asdiscussed in detail in Example 2, below.

FIG. 21 illustrates generation of a monoclonal T-cell line stablyexpressing inducible assay elements: FIG. 21A (upper left) schematicallyillustrates exemplary constructs utilized to generate infectiousparticles for the transduction of SupT1 cells with the various assayelements; FIG. 21B (right) illustrates data from a cell sorting assay;FIG. 21C (lower left) illustrates images of fluorescence microscopy ofSupT1 clones expressing the assay elements; as discussed in detail inExample 2, below.

FIG. 22 graphically illustrates data determining the optimal conditionsfor activating the assay to screen for PR inhibition: FIG. 22Agraphically illustrates a doxycycline titration using clonal SupT1 cellsharboring an inducible Gal4, PR/Gal4 or PRm/Gal4 pre-incubated witheither DMSO or 10 μM Indinavir and then either left untreated, oractivated with 50, 100, 200, 500, 1000, or 20000 ng/mL of Dox; FIG. 22Bgraphically illustrates the time course of eGFP Induction in response toDoxycyline activation in the presence of DMSO or a PI; as discussed indetail in Example 2, below.

FIG. 23 graphically illustrates data from an assay response to existingPI's; as discussed in detail in Example 2, below.

FIG. 24 schematically illustrates an exemplary assay of the invention, aconditional Protease/Gal4 fusion-based system, where GFP is activatedonly in the presence of a Protease Inhibitor: FIG. 24A schematicallyillustrates: No doxycycline, Gal4 (DB and TA domains) cannot beexpressed; FIG. 24B schematically illustrates: In the presence ofdoxycycline: D, rtTA binds to the tet-responsive element (TRE) andinduces Gal4 expression resulting in the activation of GFP expression;FIG. 24C

schematically illustrates: protease/Gal4 is expressed; however, itscatalytic activity results in the separation of the Gal4 domains,resulting in the lack of GFP expression; FIG. 24D schematicallyillustrates: in the presence of a protease inhibitor (PI), the PR/Gal4fusion remains intact, resulting in the induction of GFP expression; asdiscussed in detail in Example 3, below.

FIG. 25 schematically illustrates exemplary plasmids of the invention;as discussed in detail in Example 3, below.

FIG. 26 illustrates data from a fluorescent cell sorting assay, a FACS,showing that GFP is expressed only with the addition of an active PI; asdiscussed in detail in Example 3, below.

FIG. 27 graphically summarizes the data analysis for 24 h posttransfection: FIG. 27A. Fluorescence microscopy; FIG. 27B. Flowcytometry; FIG. 27C Quantification of Flow data; PI=10 μM Indinavir; asdiscussed in detail in Example 3, below.

FIG. 28 illustrates data: the y axis is: % GFP+ cells; the x axis is+FRGFP; for each of the paired columns the left column is control andthe right column is with inhibitor; the first lane (no columns) is mockrun; the second column pair is negative control; the third column pairis “pGal4”; the fourth column pair is “pPRm/Gal4”; and the fifth columnpair is “pPR/Gal4”; as discussed in detail in Example 3, below.

FIG. 29 illustrates constructs used for the generation of retroviralparticles; as discussed in detail in Example 3, below.

FIG. 30 illustrates plasmids for production of retroviral infectiousparticles; as discussed in detail in Example 3, below.

FIG. 31 illustrates data from a cell sorting assay where clones werescreened for the highest responsiveness to Dox and PI; where the Gal 4row shows that Tet inducible activation is very tight; the pPRm rowshows that the mutant is inactive; the PR row shows that PR/Gal4 clonesexhibit ˜90% activation with <1% background. Dox=1 μg/mL Doxycycline;PI=10 μM Indinavir; as discussed in detail in Example 3, below.

FIG. 32 graphically illustrates GFP expression in selected clones, wherethe data demonstrates that stable T-cell clones robustly report PRInhibition; as discussed in detail in Example 3, below.

FIG. 33 graphically illustrates Doxycycline Titration—pre-incubation ofclonal SupT1 cells with DMSO or 10 μM PI (Indinavir), where the datademonstrates that activation is saturated around 1 μg/mL; as discussedin detail in Example 3, below.

FIG. 34 graphically illustrates the Time Kinetics of the assay in96-well plates: Pre-incubation of clonal SupT1 cells with 10 μM PI(Indinavir)—control is no Dox with test=Activation at 1 mg/mL Dox, wherethe data demonstrates that activation of clones reaches max around 48hrs; as discussed in detail in Example 3, below.

FIG. 35 graphically illustrates data from incubating clonal T-cell lineswith various concentrations of PI's; as discussed in detail in Example3, below.

FIG. 36 schematically illustrates the HIV-1 genome and proteome, and therole of furin, PC-1 and similar host peptidases—the enzymes targeted forinhibition by assays of this invention; as discussed in detail inExample 4, below.

FIG. 37 schematically illustrates an exemplary assay of the invention;as discussed in detail in Example 4, below.

FIG. 38 schematically illustrates constructs for assays of theinvention; as discussed in detail in Example 4, below.

FIG. 39 graphically illustrates flow cytometry data from a FLAGdetection assay of the invention; as discussed in detail in Example 4,below.

FIG. 40 schematically illustrates an exemplary screening process for anassay of the invention; as discussed in detail in Example 4, below.

FIG. 41 schematically illustrates construction of a random peptidelibrary used in an alternative embodiment of the invention; as discussedin detail in Example 4, below.

FIG. 42A illustrates exemplary library inserts to corroborate quality,clones were sequenced to confirm their randomness; FIG. 42B illustratesan electrophoresis analysis; as discussed in detail in Example 4, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides methods and compositions, including chimericrecombinant proteins, nucleic acids that encode them, and cells and kitscomprising them, to screen for compositions, e.g., small molecule drugs,that can modulate, e.g., inhibit or enhance, viral proteases, includingretroviral (e.g., HIV) proteases.

In one embodiment, the invention provides cells and cell-based assaysfor monitoring the activity of viral proteases, e.g., HIV-1 protease(PR), which is an aspartyl protease. In one embodiment, these cells andcell-based assays are used to screen for and identify novel viralprotease, e.g., PR, inhibitors. In one embodiment, assays of theinvention effectively couple the surface (extracellular) expression of aprotein used as a scaffold (a scaffold protein), with the activity ofthe viral protease, e.g., PR. In alternative embodiments, polypeptidesof the invention comprise HIV p2, p7 or both p2 and p7; p2/p7 of theHIV-1 strain HXB2 (taken as the prototype genome) is part of thatvirus's viral proteome, and contains one of the natural targets for therecognition and cleavage site of PR.

In one embodiment, the scaffold is engineered for its conditionalexpression on the surface of a cell, e.g., a eukaryotic, a yeast or amammalian cell. For that purpose, in one embodiment, the scaffold isfused to a signal sequence to enable efficient and/or directedtransport, and a transmembrane domain (e.g., an Lyt2, the murine CD8molecule, and the like) is used to enable subsequent insertion in thecell membrane. In one embodiment, a tag such as a FLAG tag is added tothe scaffold downstream of the signal sequence for detection, e.g., forantibody detection, e.g., through flow cytometry or equivalentvisualization.

In one embodiment, the assay co-expresses both the scaffold protein andthe viral protease, e.g., the HIV-1 PR, which if active will bind to andcleave the scaffold at the protease recognition sequence.

In one embodiment, both scaffold and protease are co-expressed in Tcells, e.g., SupT1 T-cells, in an inducible off/on-based vector system(e.g., activated upon addition of tetracycline or doxycycline).Inducible expression of protease, e.g., PR, helps avoid its possiblecytopathic effects. Inducible expression of the scaffold is necessary asprotease, e.g., PR, will only be able to prevent surface expression ofnewly synthesized intact scaffold, as the pre-inserted scaffold wouldnot be removed from the cell surface by protease.

In one embodiment, the logic behind the engineering of the scaffold as amembrane-expressed protein is as follows: in the presence of the activeviral protease, the proteolytic enzyme will cleave the scaffold,resulting in the loss of transmembrane domain, thus preventing tag cellsurface expression. In the absence of protease, or when protease isblocked or inhibited, the scaffold will be intact and incorporated intothe membrane. As a result, the surface expression of the scaffold can bedetermined by flow cytometry allowing the discrimination between activeand inactive or blocked protease. The assay is cell-based, and can beeasily implemented for a high throughput screen, e.g., FACS. As such,the assay is invaluable for drug discovery, and can be utilized inbiological screens aimed at finding novel protease inhibitors throughrandom peptide libraries or chemical compounds libraries.

FIG. 1 is an illustration that is a representation of an exemplary assayof the invention that will enable discrimination between cleavage (leftpanel) and no cleavage (right panel) by HIV PR. SS: Signal sequence, TM:Transmembrane domain, PR: protease, orange box: proteaserecognition/cleavage site.

In one embodiment, the invention engineers a protein scaffold bearingthe protease cleavage site on the cell surface of a mammalian cell. Inone embodiment, the invention expresses, or co-expresses, a protease,e.g., a HIV-1 protease, and a scaffold used as a target, in an induciblemanner (the protease, the scaffold, or both can be expressed via aninducible mechanism, e.g., an inducible transcriptional regulator).

In one embodiment, the invention provides assays that can be adapted fora high throughput manner using e.g. flow cytometry such as FACS, and candiscriminate between active and non-active or blocked protease. In oneembodiment, the invention provides assays that can be easily adapted forhigh throughput screening. In one embodiment, the invention providesassays of this invention can be used to screen for novel proteaseinhibitors.

In one embodiment, the invention provides assays of this inventionadapted for the screen of random peptide libraries or chemical compoundsfor drug discovery.

In one embodiment, the methods of the invention use a random peptidelibrary or any peptide of choice, which can be introduced ‘in cis’,replacing the p2/p7 recognition/cleavage site, enabling the discovery ofhigher affinity sites for PR, which can be the basis for the developmentof competitor peptidomimetic drugs. In one embodiment, the randompeptide library is expressed ‘in trans’, enabling the discovery ofcompetitors/inhibitors for PR, which can be the basis for peptidomimeticdrugs.

In one embodiment, the non-biased approach of the invention permits therescue of peptides or chemicals targeted not necessarily to thecatalytic site of PR. Thus, the assays of the invention provide forextensive characterization of PR, facilitating the elucidation ofinteractions of PR with cellular targets, its mode of action andmodulation, in the context of the host cell. Assays of this inventionwill permit the replacement of PR with PR from different viral strainsor clades, or truncated versions of PR, enabling further dissection ofPR activity, and study its modulation through co-expression of cellularfactors or addition of drugs.

The assays of this invention can be further adapted to proteases ofdifferent viruses such as Hepatitis C by just exchanging therecognition/cleavage site segment of the scaffold. The assays of thisinvention can thus be exploited for the search for protease inhibitorsagainst any of the known viral pathogens that utilize their ownprotease/s as part of their lifecycles.

The assays of this invention can be adapted for the search of HIVenvelope processing inhibitors. One of the HIV proteins, envelope, isprocessed by furin and other cellular convertases. By just exchangingthe recognition/cleavage segment of the scaffold with the enveloperecognition site, the assay can be further utilized for the finding ofenvelope processing inhibitors. This same scaffold is useful for thesearch of transport inhibitors, as envelope is transported through theER, trans-Golgi network in order to be inserted within the cellmembrane.

In alternative embodiments, the assays of this invention compriseexpression of a scaffold naturally expressed in the cytoplasm that isable to be exported into the cell membrane.

In alternative embodiments, assays of this invention comprise expressionof both PR and scaffold in an off/on system for inducible expression.

In alternative embodiments, assays of this invention comprise expressionof a protein that is expressed on the surface of the mammalian cell onlywhen not cleaved by a protease, e.g., an HIV protease.

In alternative embodiments, assays of this invention can be adapted forthe screen of random peptide libraries or chemical compounds.

In alternative embodiments, assays of this invention can be implementedin mammalian cells and other cells, e.g., yeast or bacterial cells.

In alternative embodiments, methods provide for the construction of thescaffold and its expression on the cell surface. In alternativeembodiments, the p2/p7 scaffold has been engineered as described andeffectively expressed on the cell surface. In alternative embodiments,the scaffold has been introduced in a retroviral vector.

In one study, for proof of principle, the expression of an exemplaryscaffold correlates with the Green Fluorescent Protein (GFP) expression,as the vector (illustrated in FIG. 2A) contains an internal ribosomeentry site followed by GFP. The p2/p7-engineered scaffold with the FLAGtag and a signal sequence was introduced upstream of the internalribosome entry site (IRES) GFP cassette of the retroviral vectorpBMN-IRES-eGFP (illustrated in FIG. 2A) (kindly provided by Garry Nolanfrom Stanford University). The scaffold construct has been introduced ina Tet-off/on vector for inducible expression upon addition ofdoxycycline.

This construct allows stable expression of the insert and correlation ofgreen fluorescence with expression of the gene of interest (p2/p7 in ourcase). This vector was used to corroborate the expression of a scaffoldof this invention on the cell surface by flow cytometry, as graphicallyillustrated in FIG. 2B: a FACS analysis of 293T and 293T-FLAG-p2/p7expressing cells. The FLAG-p2/p7 scaffold was introduced upstream anIRES-GFP cassette in the pBMN-IRES-eGFP retroviral vector (asillustrated in FIG. 2A). Cells were stained with anti-FLAG (Sigma) andAPC-coupled secondary antibody (Invitrogen, Carlsbad, Calif.) (rightlower panel). SS: Signal Sequence, TM: Transmembrane domain.

The Expression of Protease in a Non-Toxic Inducible Manner.

In one embodiment, to achieve low levels of protease, e.g., PR,expression in mammalian cells, a tetracycline (Tet) inducible system isused. FIG. 3 illustrates by fluorescence microscopy use of an exemplaryvector of the invention, a Tet-inducible HIV-based self-inactivatingvector, that allows regulated expression of the gene of interest. Inthis case, we have used the enhanced green fluorescent protein (eGFP) asthe ectopic gene. This vector allows different levels of proteinexpression. Tight repression and expression of PR at low levels may becrucial to avoid the possible side effects of PR. It is important tomention that this system is an off/on system that allows for expressionof the gene of interest only upon addition of tetracycline ordoxycycline. The inducible system allows for de novo synthesis of thescaffold, needed for the successful implementation of the assay. FIG. 3illustrates by fluorescence microscopy HeLa cells infected with a Tetinducible HIV-based self-inactivating vector. The cells were incubatedwith 1 μg/ml Tet and eGFP expression was observed by fluorescencemicroscopy 24 and 72 h post addition of Tet. PR was expressed by itselfand as a GFP fusion. As high level expression of PR might be toxic tothe cell, the PR inhibitor Saquinavir was added to inhibit its activitybut not its expression.

As expected, the N-terminal GFP fusion is highly expressed in this case,as illustrated in FIG. 4, lower panels. Importantly, this fusion seemsto be expressed at low levels even in the absence of inhibitor (asillustrated in FIG. 4, upper panels and FIG. 5), demonstrating thatactive PR can be expressed at low levels without cytotoxic effects. Insummary, FIG. 4 illustrates fluorescent microscopy analysis of cellstransfected with pcDNA control (Invitrogen) and pcDNA-GFP-PR. GFPexpression was analyzed 24 h post transfection. FIG. 4 Upper panels:untreated cells, FIG. 4 Lower panels: PR inhibitor, Saquinavir (NIHReagents Program) was added at 0 and 24 h post transfection.

In summary, FIG. 5 graphically illustrates flow cytometry analysis ofcells expressing PR-GFP fusion. Cells transfected with pcDNA (control)or pcDNA-GFP-PR fusions were collected 48 hr post-transfection andanalyzed for GFP expression. FIG. 5 Upper panels: untreated cells. FIG.5 Lower panels: Cells treated with Saquinavir.

In order to prove that those cells are actually expressing active PR,cells transfected with the pcDNA control or the N-terminal GFP fusion;GFP-PR were collected for Western blot analysis with anti-PR antibody(NIH Reagents Program). The presence of degradation products ofautolysis when no inhibitors are added demonstrates that PR retains itsactivity, see FIG. 6. FIG. 6 illustrates a Western blot analysis ofprotease (PR) activity. 293T cells transfected with pcDNA (control) orpcDNA-GFP-PR fusions and either untreated or treated with Saquinavir (3μM/ml) (e.g., INVIRASE™ or FORTOVASE™) at 0 h and 24 hrpost-transfection) were collected for the blots. The expected autolysisproducts are shown. Upper panel: anti-PR antibody (NIH AIDS reagent).Lower panel: anti-alpha-actin antibody (Invitrogen, Carlsbad, Calif.) asloading control).

This exemplary scaffold of the invention, an adapted scaffold, is basedon the same idea, but with an important difference. In one embodiment,when protease (e.g., PR) is active the FLAG or other detectable tag willbe present on the surface and detectable e.g., by flow cytometry,whereas when protease (e.g., PR) is blocked or inactive, the FLAG willbe lost and not expressed on the cell surface. This scaffold is based onthe idea that generally, proteins to be expressed on the surface of thecell have a signal sequence (SS) on their N terminus that targets themto the endoplasmic reticulum (ER) and a transmembrane domain (TM) thatretains them in the membrane.

On the other hand, proteins that are retained in the ER, will have, inaddition to the SS and the TM, an ER-retention signal such as theprototypic KDEL sequence. This sequence is known to have strong affinityto the KDEL receptor (SEQ ID NO:1), acing the luminal side of the ER. Inone embodiment, the ER retention motif or KDEL (SEQ ID NO:1) motif ispositioned in the scaffold protein such that when PR is active thescaffold will be separated into two pieces, leaving the ER retentionmotif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of thepolypeptide in the ER and freeing the detectable moiety-comprisingportion to the cell's extracellular membrane, and if PR is blocked orinactive, the entire scaffold polypeptide will be retained in the ER,and as a consequence will not be detected on the cell's extracellularsurface

FIG. 7 illustrates an exemplary construct of the invention that is basedon CCR5 but has only the two first TM domains (rather than the originalseven). As shown in the figure, the scaffold is further fused to a KDELsequence at its C-terminus This scaffold allows, as mentioned earlier,to introduce a recognition sequence in the loop facing the lumen (forpeptidases such as furin), or in the loop facing the cytoplasm (forviral proteases). FIG. 7 illustrates shows an exemplary scenario formonitoring of protease, e.g., PR, activity. When protease, e.g., PR, isactive (left panel of FIG. 7), the scaffold will be separated into twopieces, leaving the KDEL portion in the ER and freeing the FLAG tagportion to the membrane, where it will be detected by flow cytometry. Ifprotease, e.g., PR, is blocked or inactive (right panel of FIG. 7), theentire scaffold will be retained in the ER, and as a consequence willnot be detected on the surface.

For example, the exemplary CCR5 engineered protein (or partial CCR5 asdescribed here and in the figure) can be replaced by any other proteinof choice or hybrid protein. For example, one exemplary embodimentcomprises a hybrid protein comprising the N-terminus of the CD8 moleculeor the CD8 molecule equivalent in mice (referred to as Lyt2), comprisingits natural SS, and the C-terminus of the chemokine receptor CCR5including only the last TM domain. In this embodiment, only requirementis that the resulting protein will, when cleaved, retain theKDEL-containing side in the ER and the N-terminus on the cell surface.

In alternative embodiments, any protein is used as scaffold (instead ofthe one exemplary protein described herein), provided that by adding aKDEL sequence at the C terminus the polypeptide will be retained it inthe ER, unless separated from the N-terminus.

In this embodiment, a p2/p7 recognition site is imbedded in thecytoplasmic loop of the scaffold, as PR is known to be active in thisenvironment.

In alternative embodiments, the scaffold is engineered for the search ofviral PR inhibitors (active in the cytoplasm) and/or forproteases/peptidases active in the lumen of the ER. In one embodiment,the loop facing the luminal face of ER is substituted by a recognitionsite cleaved by cellular peptidases. This can include the gp120/gp41boundary, known to be cleaved by peptidases such as furin. These enzymesare known to be active in the inner side of the ER, that is its luminalface, making this exemplary scaffold adaptable for the search of HIVenvelope processing and for transport inhibitors.

In alternative embodiments, the invention provides methods andcompositions, including chimeric recombinant proteins, nucleic acidsthat encode them, and cells and kits comprising them, to screen forcompositions, e.g., small molecule drugs, that can modulate, e.g.,inhibit or enhance, any enzyme, e.g., protease or HIV-1 protease (PR),or NS2/NS3 or NS3/NS4A protease of HCV, or any viral protease, includingretroviral (e.g., HIV) proteases, and/or any transport and/or structuralprotein.

In one embodiment, the invention provides cells and cell-based assaysfor monitoring the activity of activity an HIV-1 protease (PR), which isan aspartyl protease. In one embodiment, these cells and cell-basedassays are used to screen for and identify novel PR inhibitors. In oneembodiment, assays of the invention effectively couple the surface(extracellular) expression of a protein used as a scaffold (a scaffoldprotein), with the activity of the viral PR. p2/p7 of the HIV-1 strainHXB2 (taken as the prototype genome) is part of that virus's viralproteome, and contains one of the natural targets for the recognitionand cleavage site of PR.

In one embodiment, the scaffold is engineered for its conditionalexpression on the surface of a cell, e.g., a yeast or a mammalian cell.For that purpose, in one embodiment, the scaffold is fused to a signalsequence to enable efficient transport, and a transmembrane domain(e.g., an Lyt2, the murine CD8 molecule, and the like) is used to enablesubsequent insertion in the cell membrane. A tag such as a FLAG tag isadded to the scaffold downstream of the signal sequence for detection,e.g., for antibody detection, e.g., through flow cytometry or equivalentvisualization.

In one embodiment, the assay co-expresses both the scaffold protein andthe HIV-1 PR, which, if active, will bind to and cleave the scaffold.

In one aspect, both scaffold and protease are co-expressed in alymphocyte, e.g., a T cell or T cells, e.g., a SupT1 T-cell, in aninducible off/on-based vector system (e.g., activated upon addition oftetracycline or doxycycline). Inducible expression of PR helps avoid itspossible cytopathic effects. Inducible expression of the scaffold isnecessary as PR will only be able to prevent surface expression of newlysynthesized intact scaffold, as the pre-inserted scaffold would not beremoved from the cell surface by PR.

Kits

The invention provides kits comprising compositions and instructions foruse of the invention. The kits can include: cells comprising nucleicacids encoding the chimeric polypeptides of the invention (the “scaffoldproteins”) and/or vectors comprising these nucleic acids, or chimericpolypeptides of the invention, transfecting agents, transducing agents,instructions (regarding the methods of the invention), or anycombination thereof. As such, kits, cells, and libraries of compoundsare provided herein.

Cell-Based Methods and Multiplexed Systems

In alternative embodiments, the invention provides cells and cell-basedassays and multiplexed systems for monitoring the activity of activityof proteases, e.g., an HIV-1 protease (PR), which is an aspartylprotease. In one embodiment, these cells and cell-based assays are usedto screen for and identify novel PR inhibitors (“PIs”). In oneembodiment, the invention provides methods and compositions, includingchimeric recombinant proteins, nucleic acids that encode them, and cellsand kits comprising them, to screen for compositions, e.g., smallmolecule drugs, that can modulate, e.g., inhibit or enhance, any enzyme,e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A proteaseof HCV, or any viral protease, including retroviral (e.g., HIV)proteases.

In one embodiment, the invention provides assays and multiplexed systemsin T cells to monitor the proteolytic activity of a protease, e.g., theHIV-1 protease (PR). The assay is based on an inducible Gal4 HIV-1 PRfusion which binds to upstream activation sequences and activates areporter gene only in the presence of a PR inhibitor (“PI”). The assaywas developed through retroviral technology in T-cells to mimic thenatural environment of HIV infection.

In one embodiment, the invention provides clones which, when activated,express eGFP as a biosensor of PR activity. This assay of the inventionhas a robust and reliable readout that relies on green fluorescence,making it ideal for high-throughput screening utilizing flow cytometry.Thus, the assay of the invention will greatly facilitate the search fornovel peptide- and chemical-compound-based PIs in T-cells.

In one embodiment, the invention provides a simple, rapid andstraightforward method and multiplexed systems for monitoring aprotease, e.g., PR, activity to facilitate the search for novelinhibitors/competitors of the protease that could lead to newtherapeutics, e.g., to treat HIV (e.g., AIDS).

In one embodiment, assays of the invention are based on the classicalGal4-UAS system, a broadly utilized system for the analysis of geneexpression. The yeast Gal4 protein represents a prototypic transcriptionfactor consisting of two separate domains: An N-terminal DNA-bindingdomain (DBD: aa 1-147) and a C-terminal Transactivation domain (TAD: aa768-881)'. The Gal4 protein binds to consensus Upstream ActivationSequences (UAS's) via its DBD and activates transcription of downstreamgenes through its TAD. However, when the two Gal4 domains are separated,neither half of the protein can independently serve as a functionaltranscription factor.

Murray (1993) Gene 134(1):123-128, demonstrated the ability for HIV-1 PRfused within Gal4 to auto-catalytically remove itself, leaving behindthe two non-functional domains of Gal4. When the PR/Gal4 fusion proteinis mutated at the catalytic site, however, or is in the presence of aninhibitor, the fusion protein remains intact, retaining its ability tobind to UAS through the DBD and activate transcription through TAD. Inalternative embodiments, this property is incorporated into thisinvention to express a reporter gene in an inversely proportional mannerto PR activity and serve as template for this assay.

In alternative embodiments, assays and multiplexed systems of thisinvention are based on the expression of the PR/Gal4 fusion as aninducible fusion through a Tet-On system (e.g., in one embodiment,adapted from Clontech, Takara Bio Inc., Shiga, Japan), thus drasticallyreducing its possible toxic side effects. In this embodiment, thereverse tetracycline transactivator (rtTA) is utilized, allowing for theinduction of PR/Gal4 expression only upon addition of tetracycline (Tet)or doxycycline (Dox). The readout; eGFP expression, will appear onlywhen PR/Gal4 expression is induced in the presence of inhibitor.Moreover, all the elements of the assay have been constructed inretroviral vectors for their stable expression in mammalian cells. Inalternative embodiments, the assays of the invention are designed foruse in lymphocytes such as T cells to facilitate the high-throughputscreening for novel inhibitors in a more natural milieu.

In alternative embodiments, assays of the invention are adapted suchthat the cells carry several enzyme, e.g., protease or HIV-1 protease(PR), or NS2/NS3 or NS3/NS4A protease of HCV, mutant variants, includingfor example the most prevalent PR mutant shown to be resistant toFDA-approved inhibitors. In alternative embodiments, clones comprisingdifferent enzymes (e.g., PRs), when inhibited, activate thetranscription of a different fluorescent marker. Accordingly, inalternative embodiments, the assays of the invention are adapted asmultiplexed systems.

We have proved the clones of this invention to be very valuable for thescreening of inhibitors against the specific PR used in the assay; fromthe HXB2 consensus T-tropic strain. Due to the high mutational rate itis crucial to adapt the assay to as many protease variants aspossible—and the assays and multiplexed systems of the invention areadaptable to multiple protease variants. In alternative embodiments,assays of the invention are adapted to an array of proteases thatinclude the most prevalent protease variants resistant to the existingFDA-approved PIs, In alternative embodiments, these assays areconfigured as multiplexed systems of the invention.

In alternative embodiments, assays of the invention are adapted tomutations in at least 18 different positions within the 90 amino-acidsof PR which have been described to confer drug resistance. In order toadapt the assay for multiplex analysis, we chose the three mostprominent variants: L90M, 154V and V82A. For this purpose, inalternative embodiments, L90M, 154V and V82A are introduced between theGal4 DBD and TA domains. In alternative embodiments, each PR variantwhen inhibited will activate a different fluorescent protein. While thewild-type variant described in FIG. 6 data activates e-green fluorescentprotein, or eGFP (excited with the 488 nm blue laser, the other mutantswill activate an e-cyan fluorescent protein (or eCFP, using a 405 nmviolet laser), and mOrange or mCherry (561 nm yellow laser). This willallow screening for compounds that inhibit all, some or one PR variantat a time, based on the specific fluorescence observed. Importantly,other clones expressing a different set of mutant PR will be produced inthe future, when necessary.

In alternative embodiments, assays of the invention are adapted tomultiplexed formats with various enzyme (e.g., PR) mutants/variants andreporter combinations to simultaneously detect enzyme (e.g., PR)resistance to individual hits. In alternative embodiments, assays of theinvention are adapted to Luminescence/plate reader-based formats. Inalternative embodiments, assays of the invention are adapted toscreening peptide and chemical-compound libraries.

Cell-Based Assays for the Identification of Compositions that InhibitEnvelop Processing

In alternative embodiments, the invention provides cells and cell-basedassays to screen for compositions, e.g., small molecules or drugs, thatinhibit or modify the activity of enzymes such as calcium-dependentprotein convertases such as furin involved in HIV envelop proteinprocessing, including cleavage of the HIV gp160 envelope precursor,resulting in gp120 and gp41 envelope products.

In one embodiment, the invention provides assays to monitor the HIV-1envelope processing process. This process is based on the cleavage ofthe gp160 envelope precursor, resulting in gp120 and gp41 envelopeproducts. In one embodiment, the assay of the invention is based on theengineering of a receptor protein scaffold (or detection moiety)construct that will be targeted to the ER and transported to the cellsurface only when the protein scaffold was efficiently cleaved by agp160 envelope precursor processing enzyme such as a calcium-dependentprotein convertase, e.g., prohormone convertase-1 (“PC1”), furin and/orsimilar enzymes (e.g., any member of the subtilisin/kexin family ofproprotein convertases), some of which reside in the ER-trans Golginetwork.

In one embodiment, this is attained by fusing a KDEL (SEQ ID NO:1)retention signal, known to be recognized and bound by KDEL-receptors inthe ER lumen, at the carboxy-terminus (C-terminus) of the scaffold (ordetection moiety). An enzyme (e.g., furin, PC1, a member of thesubtilisin/kexin family of proprotein convertases, and the like)recognition/cleavage site is introduced between the scaffold and theKDEL (SEQ ID NO:1) sequence. When enzyme (e.g., furin, PC1, etc.)processing is blocked or inhibited, the receptor will move from the ERto the trans-Golgi and recycled back to the ER, due to the presence ofthe KDEL (SEQ ID NO:1) sequence. In contrast, when the enzyme (e.g.,furin, PC1, etc.) is active, it will cleave its recognition site andseparate the KDEL (SEQ ID NO:1) sequence from the scaffold protein,which will then be allowed to travel to the surface.

In one embodiment, assays of the invention facilitate the monitoring ofenzyme (e.g., furin, PC1, etc.) activity based on the presence orabsence of the scaffold (including any detection moiety) on the surfaceof the cell. In alternative embodiments, fluorescent-coupled antibodiesagainst a tag (e.g., FLAG in our example) can be used to analyze cellsby flow cytometry or similar or equivalent detection system.

Assays of the invention can greatly facilitate the discovery of novelgp160 processing inhibitors, including screening for any composition,including a small molecule, protein, carbohydrate and the like that canact as a partial or complete gp160 processing inhibitor.

In one embodiment, assays of the invention are T-cell-based; in thisembodiment the assay represents the natural milieu for HIV, e.g., HIV-1,infection.

In one embodiment, flow cytometry allows utilization of assays of thisinvention in a high-throughput manner. Thus, in alternative embodiments,assays of the invention can be used for the screening of chemicalcompound (e.g., small molecule) libraries aimed at finding novelinhibitors of gp160 processing. In one embodiment, the nature of theassay as cell-based will discriminate between drugs that target Furin(which would be detrimental to the cell), and those that target thespecific cleavage of the gp120/41 boundary by Furin (or similarenzymes).

In one embodiment, the CCR5 receptor, a naturally present receptor inmacrophages and other cell types, is engineered as a scaffold (e.g.,detection moiety) to satisfy the needs of this assay. In this exemplaryembodiment, the scaffold will be comprised of two transmembrane domains(TMs) of the CCR5 receptor fused to a FLAG molecule on the N-terminalregion. We have chosen TM1 and TM2 from the original seven TMs of CRR5,but any TM could be used instead. The KDEL (SEQ ID NO:1) ER retentionsignal will be fused to the C-terminus in order to keep the scaffoldbound to the ER membrane, at the luminal face.

In one embodiment, the gp120/gp41 boundary, including therecognition/cleavage site REKRA (SEQ ID NO:14) (amino-acids 515-519) andseven additional amino-acids at both sides (AKRRVVQREKR (SEQ ID NO:15)AVGIGALF (SEQ ID NO:16), which represents amino-acids 502-519 of theHXB2 HIV-1 strain), are introduced into this exemplary chimeric proteinof the invention.

While the invention is not limited by any particular mechanism ofaction, if cellular proteases/peptidases, such as Furin or PC1, residentin the ER lumen, cleave the scaffold, the receptor will travel to thecell surface and be recognized with a detection system, e.g., a flowcytometry. If, in contrast, the protein is not cleaved, the scaffoldlevel on the cell surface will be extremely diminished or completelyabolished. As a control, a similar scaffold that lacks the cleavage sitecan be used to ensure expression in the ER but not on the surface. Theintroduction of the gp120/41 boundary can be performed with the additionof restriction enzyme cleavage sites on both sides of the sequence,allowing for easy replacement of other sequences in the future. Thiswill also facilitate the exploration of other consensus sequences foundto be recognized by Furin, or similar enzymes, such as the sequencewithin the V3 loop of gp120.

Though HIV envelope is known to be cleaved in the trans Golgi network,in one embodiment the scaffold is localized to the ER through the KDEL(SEQ ID NO:1) signal because the mechanism KDEL receptor-bound proteinsare known to travel to the Golgi and subsequently recycled into to theER.

While one exemplary embodiment comprises a protein scaffold based onCCR5, CCR5 was chosen only as a proof of concept—in alternativeembodiments any protein, mimetic, peptidomimetic or equivalent scaffoldcan be used.

In alternative embodiments the assays of the invention are adapted tovirtually any construct that behaves as described herein, and thuscomprises, from N- to C-terminus:

-   -   a signal sequence for ER targeting,    -   a tag for antibody recognition,    -   two transmembrane domains that will span the ER membrane, with        an extra loop at the ER luminal face    -   a Furin or similar enzyme recognition/cleavage site spanning a        segment of the gp120/41 boundary, facing the ER lumen    -   a KDEL (SEQ ID NO:1) sequence for ER retention.

While one exemplary embodiment comprises introduction of only a shortsequence of the gp120/gp41 boundary that contains the consensus Furinrecognition/cleavage site (ten amino-acids in length), in alternativeembodiments additional recognition sites of different lengths could beintroduced instead. In alternative embodiments assays of the inventionuse two known consensus sequences of the HXB2 HIV strains gp120.gp41boundary sites: AKRRVVQREKRAVGIGALF (SEQ ID NO:17) (recognition/cleavagesite in bold). Other strains are used as well.

The different scaffolds used in various embodiments of this inventioncan further our understanding of protein transport through the ER to thecell surface.

In alternative embodiments the cell-based assays are used in T-cellsthrough retroviral technology. T-cells are a cell type readily infectedby HIV, thus providing an art-accepted model that mimics naturalinfection. In alternative embodiments, the assay is used in lymphocytes,e.g., T-cells—a cell type readily infected by HIV through retroviraltechnology.

In alternative embodiments these cell-based assays facilitate thediscovery of novel drugs aimed at HIV-1-envelope processing rather thanthe host protease, thus avoiding toxic side-effects, e.g., avoidingcytotoxic side-effects.

In alternative embodiments, assays of the invention monitor HIV-1envelope processing based on the cleavage of the gp160 envelope proteinprecursor, resulting in gp120 and gp41 products using a scaffold proteinthat will not be retained on the cell-surface when processing is blockedor inhibited. In alternative embodiments, the processing, or inhibitionof processing, is detected by flow cytometry allowing the observer todiscriminate between active and inactive envelope processing.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Exemplary Assays of the Invention

The invention provides compositions and assays for screening forprotease inhibitors, e.g., viral protease inhibitors such as HIV-1protease (PR) (an aspartyl protease) inhibitors. PR is required for theefficient processing of the Gag and Gag-Pol precursor polyproteins; acritical step in the viral life cycle. In alternative embodiments, theinvention provides compositions and assays for: (1) Discerning theeffects of protease, e.g., PR, on signaling cascades of the host cell,and (2) Developing novel cell-based assays to enable screening ofpeptide libraries for the search of novel protease, e.g., PR,inhibitors. In alternative embodiments, a protease, e.g., PR, isexpressed as a fusion protein in the presence of limiting levels ofinhibitors, in different cellular compartments and in an induciblemanner.

FIG. 8 illustrates HIV-1 Genome organization: HIV-1 has three structuralgenes (Gag, Pol and Env) and six regulatory genes. Both Gag and Pol,expressed as Gag-Pol precursor are cleaved by the HIV PR, while Env iscut by the cellular protease furin. PR autocatalytically cleaves itselffrom the precursor polyprotein. HIV PR therefore determines theinfectious potential of the virus, as its activity is crucial for theconversion of the immature virion to the mature, infectious form.

FIG. 9 illustrates an indirect confirmation of HIV-1 PR activity: 293Tcells were transfected with the plasmids pCMVD8.2, D8.7 and D8.91(illustrated in FIG. 9A) with and without inhibitors. Ritonavir, a knownPR inhibitor, was added to cells at a concentration of 3 mM/ml every 24hours. Cells were extracted 48 hours post-transfection and analyzed forcapsid (p24) expression. The presence of p24, detected by Western blot,as illustrated in FIG. 9B, is therefore an indicator of PR activity.

FIG. 10 illustrates an exemplary construct of the invention (LTR/MinCMV-TO/GFP/pPGK/rtTA/IRES/Puro/LTR) and the results of an inducibleexpression system of the invention: in one embodiment, for theexpression of low levels of PR in mammalian cells, a tetracycline (Tet)inducible system is used. To corroborate the efficiency of the exemplaryconstruct and system of the invention, Green Fluorescent Protein (GFP)was used as a marker for inducible expression. Fluorescence intensitywas monitored in 293T cells transfected with the Tet inducible HIV-basedself-inactivating vector that allows for the regulated expression of thegene of interest. Importantly, this exemplary vector allows for thecontrolled expression of PR, which may be essential in order to avoidPR-mediated cytotoxicity.

FIG. 11 illustrates the expression of two exemplary HIV-1 PR/GFP fusionproteins of the invention as illustrated in the schematics, and insummary:

FIG. 11A: expression of pCMV-PR/P1A/D25N-GFP=>pSV40-Zeo;

FIG. 11B: expression of pCMV-GFP-PR/P1A/D25N=>pSV40-Zeo. In order todetect PR expression, GFP was fused to the C (left panels) or N (rightpanels) terminus of PR. In addition to wild-type PR (top of panels), amutant form; PIA was also expressed in 293T in a similar fashion (Bottomof Panels). This mutation may increase PR flexibility.

FIG. 12 illustrates a Western Blot of PR/GFP fusion proteins withanti-PR antibody. A KDEL sequence was fused for further expression inthe ER (last 4 lanes).

FIG. 13 illustrates targeting HIV-1 PR to different cellularcompartments. PR was targeted to the nucleus by adding a nuclearlocalization signal to the C terminus using the exemplary vector:pCMV-PR/P1A/D25N-GFP/NLS=>pSV40-Zeo. Nuclear expression of PR will allowco-localization with one of our scaffold proteins (GFP based scaffold,see FIG. 16). Moreover, expressing PR in a different cellularcompartment may decrease its cytotoxic effects without affecting itscatalytic activity.

FIG. 14 illustrates a schematic representation of an exemplary scaffoldof the invention and an exemplary protease (e.g., PR) assay based onsurface expression of the scaffold protein. The scaffold(SS-FLAG/P2/P7/TM) was engineered to be expressed on the cell surfaceand consists of an HIV-1 sequence with one of the PRrecognition/cleavage sites. This cleavage site has the highest affinityto PR and is the first one to be cleaved. The design of the scaffoldallows for surface expression only when protease (e.g., PR) is inactiveor inhibited. Surface expression of the scaffold can then be easilyidentified by flow cytometry. This assay serves as a platform for highthroughput screening of peptide libraries targeted against protease(e.g., PR).

FIG. 15 illustrates two exemplary constructs of the invention:pCMV/GFP=>Zeo, and pCMV/GFP=>SS-FLAG/P2-P7/TM=>Zeo; and flow cytometryanalysis of the expression of these two scaffold proteins on the surfaceof 293T cells. Cells were transfected with either a control GFP vector(pCMV/GFP=>Zeo) or a scaffold protein construct of the invention(pCMV/GFP=>SS-FLAG/P2-P7/TM=>Zeo). Cells were then stained withanti-FLAG-PE antibody and analyzed by flow cytometry; FIG. 15Aillustrates results for the control construct and FIG. 15B illustratesresults for the scaffold protein construct. The double-positivepopulation expressing the scaffold can be enriched through sorting.

FIG. 16 illustrates the results of use of an exemplary construct of theinvention, pCMV/GFP/PCS/GFP-NLS=Zeo, where GFP as a biosensor of PRactivity. In this embodiment, the surface expression scaffold assay isbased on GFP. The PR cleavage site (PCS) has been introduced into one ofthe loops of GFP. This construct was then transfected into 293T cells,corroborating that fluorescence was maintained. Thus, in thisembodiment, the GFP is expressed in the absence of PR. In contrast,cleavage by PR will result in truncation of the GFP into two halves andthus, loss of fluorescence. It has been shown that the reconstitution oftruncated GFP products can restore fluorescence. Therefore, we havefused a nuclear localization signal to the C terminal half to isolate itfrom the N terminal half, thus preventing the reconstitution offluorescence. This exemplary scaffold can serve as an indicator of PRactivity.

FIG. 17 schematically illustrates an exemplary construct of theinvention (aa1-134/protease cleavage site/aa136-238/NLS) where a nuclearlocalization signal (NLS) is separated by a detectable moiety, e.g., afluorescent protein, by a protease cleavage site, and the proteasecleavage site is spliced into the middle of the fluorescent protein;thus, if the protease (e.g., a PR) is inactive or inhibited, the NLSretains the detectable moiety in the nucleus, while an active proteasecompletely eliminates the fluorescent signal.

FIG. 18 schematically illustrates an exemplary assay of the invention.In this embodiment, a random peptide library, the scaffold protein, andPR is co-transfected into SupT1 cells. Cells can be co-transfected withPR and the scaffold protein and, in one embodiment, expressed in aninducible manner, e.g., a Tet inducible manner. Cells selected for bothPR and scaffold can be transfected with a random peptide library (e.g.,a retroviral peptide library, as in the schematic). Using FACS analysis,cells that are positive for the scaffold can be selected and cloned. Inone inducible manner, the peptide will then be rescued by PCR andreintroduced into naïve cells to corroborate its inhibitory effect. Thepeptide can then be the basis for the development of peptidomimeticanti-viral drugs.

CONCLUSIONS

In alternative embodiments, the invention provides compositions andassays to measure the activity of proteases, e.g., viral proteases, suchas HIV-1 PR, a difficult protein to study due to its instability andpossible cytotoxic effects. In alternative embodiments the inventionexpresses PR in a stable manner for a cell based assay aimed at findingpeptides that inhibit PR, as well as discerning its effect on signalingcascades. In alternative embodiments, GFP fusion proteins wereconstructed. These products allow determination of the sub-cellularlocalization of HIV-1 PR. Moreover; we have also shown that PR can betargeted to different organelles. This is important as it will enabledecreasing its toxicity, without affecting the catalytic activity of PR.

In alternative embodiments, the invention elucidates the effects of PRon target cells. In alternative embodiments the invention PR isexpressed in a Tet-inducible manner to help in elucidating the signalingcascades influenced by PR, as turning on/off the expression of PR canavoid cytotoxic effects and thus become the perfect model for studyingsignaling.

In alternative embodiments the invention provides assays for determiningPR activity because PR is one of the main targets for antiviral therapy.In alternative embodiments PR is expressed to find novel PR inhibitors.

We have shown that a scaffold bearing a PR recognition site can beefficiently expressed on the surface of mammalian cells, enabling itsdetection by flow cytometry. We have also expressed a GFP scaffold withan internal PR cleavage site. In alternative embodiments, the scaffoldsof the invention are used in high throughput screening for finding novelpeptide inhibitors against PR.

Example 2 Exemplary Assays And Multiplexed Systems of The Invention

In alternative embodiments the invention provides compositions, assaysand multiplexed systems for screening for protease, e.g., HIV-1 protease(PR) (an aspartyl protease), inhibitors. PR is required for theefficient processing of the Gag and Gag-Pol precursor polyproteins; acritical step in the viral life cycle.

Exemplary Assay overview: In order to establish a reliable andreproducible assay for monitoring the activity of HIV-1 PR, specificallydesigned to maximize throughput capabilities, it was important toaddress several issues.

First, we aimed at establishing the assay as a cell-based assay, and ina cell type that would mimic the natural environment of HIV-1 infection,such as T-cells. Secondly, because of the possible toxic side effects ofPR the assay needed to be designed in an off/on system for the inducibleexpression of PR. Finally, in order for the assay to provide significantbenefits, it needed to include a straight-forward readout such as eGFPexpression. This would allow for the analysis by flow cytometry, with nonecessary staining and make the assay easily adaptable tohigh-throughput screening. An overview of this exemplary assay of theinvention is described in FIG. 1.

This exemplary assay relies on the presence or lack of eGFP expressionthat serves as biosensor for PR activity. The assay is designed so thatcells expressing blocked or inactive PR can be easily discriminated fromthose expressing active PR based on eGFP expression.]

Confirmation of a reporter/activator (Gal4/UAS) system in mammaliancells: Before the establishment of a stable cell line expressing all ofthe elements of the assay, it was crucial to first verify that theelements of the assay respond transiently as expected. Preliminaryexperiments were performed in adherent HEK 293T cells. First, we testeda reporter vector containing a 5XUAS Gal4 responsive element upstream ofa minimal CMV promoter followed by the eGFP reporter gene. When 293Tcells were transfected with this vector alone, there was little to nobackground. This lack of background expression from the reporter genewas vital to the success of developing a reliable assay. To investigatethe level of Gal4-dependent eGFP expression, we first co-transfected thereporter vector with pcDNA3.1-Gal4, a construct based on the pcDNA3.1vector for mammalian expression (see Figures). The Gal4 gene utilizedhere encodes only for the DBD and TAD segment of Gal4. While othervariants of Gal4 (such as Gal4VP16) are capable of significantly higherinduction of genes under UAS control, minimal Gal4 allows for simpleinsertion of a proteolytic enzyme within the two distinct domains whosebehavior has been well characterized. As expected, co-transfection ofthe reporter vector with pcDNA-Gal4 led to a dramatic induction of eGFPexpression in 293T cells (see Figures).

Insertion of the HIV-1 PR sequence between the Gal4 domains does notsubstantially disrupt Gal4 activity in mammalian cells: Next, wecorroborated that the insertion of a PR sequence within the Gal4 DBD andTAD domains maintains Gal4's ability to serve as a functionaltranscription factor. To test this, we first introduced a mutatedversion of PR reported to be inactive. This fusion was designed toensure that the insertion ‘per se’ of the specific PR sequence does notjeopardize the ability of Gal4 to act as a transcription factor. Forthis purpose, the HXB2 HIV-1 sequence of the PR mutant version D25Aincluding the 22 upstream amino-acids and the 32 downstream amino-acidsof PR (to include the PR cleavage sites) was introduced in between theGal4 DBD and TAD within a pcDNA3.1 vector (Figure). PR D25A haspreviously been shown to lack catalytic activity, and, as such, shouldnot be able to separate the domains, nor disrupt the ability of DBD andTAD to work in conjunction and activate the reporter eGFP expression. Asexpected, reporter and pcDNA-PRmiGal4 co-transfection resulted insignificant eGFP expression (see Figures). Although the induction ofeGFP expression by the PRm/Gal4 fusion was less than that of Gal4 alone,the level of activation was sufficient for a clear and reliable readout.

Wildtype PR fused between the Gal4 DBD and TAD results in atranscription factor with abolished activity: We next substituted thePRm sequence with the wild-type sequence. This sequence contained theexact same additional 22-upstream and 32-downstream amino-acids from PR,but retained the wild-type aspartic acid residue at position 25.Co-transfection of the reporter vector with wild-type PR between theGal4 domains led to a significant reduction in reporter eGFP expressioncompared to the mutant fusion or Gal4 alone.

Wildtype PR fused within the Gal4 DBD and TAD has restoredtranscriptional activation in the presence of a PR inhibitor. It was ofcritical importance to verify the ability for the PR/Gal4 fusion toactivate the reporter eGFP by the addition of PR inhibitors. For thatpurpose, 293T cells were pre-treated with 10 μM Indinavir and thenco-transfected with reporter and pcDNA-PR/Gal4 vectors. While controlcells or cells incubated with 10 μM DMSO lacked eGFP expression, cellsincubated with 10 uM Indinavir showed a drastic induction in HEK293Tcells. As expected, the activity of PR/Gal4 was restored in the presenceof inhibitor, as seen by the UAS-dependent transcription of theinhibited fusion protein (see Figures).

Design of lentiviral constructs and infection of T-cells with the assayelements. We next addressed the question whether these results could bereproduced in T-cells, a cell-type that represents a more natural milieufor HIV-1 infection. For that purpose, we have utilized retroviraltechnology to stably express the elements of the assay in mammaliancells. Reporter element and Gal4 or PR/Gal4 fusions were transferredinto lentiviral vectors. First, the reporter sequence was inserted intoan HIV-based self-inactivating lentiviral vector with a modified U3sequence was utilized to ensure that no reporter background activity wasobserved in the absence of an inhibitor. Secondly, we wished to createinducible expression of PR/Gal4 fusions. This would alleviate ourconcern for the difficulty in creating a stable cell line expressing PRdue to the reported possible cytotoxicity of active PR in mammaliancells.

To obtain an inducible cell line, we utilized the tetracycline induciblesystem (Tet-On). For this purpose we constructed two lentiviral vectors;one harboring a 7× Tetracycline Response Element (TRE) upstream the geneof interest and another expressing the reverse tet-transactivator (rtTA)coupled to an IRES-mCherry cassette to corroborate rtTA expression. Inthis system, the TRE element is bound and activated by rtTA only in thepresence of an inducer (Tet or Dox). Gal4, PR/Gal4 and PRm/Gal4 were alltransferred into the TRE inducible vector (see Figures).

The UAS Reporter and PR(m)/Gal4 fusions behave similarly in SupT1 cellsas HEK293T cells. We first confirmed whether the results obtained in thetransient experiments in 293T cells with reporter and PR/Gal4 vectorscould be reproduced with the lentiviral reporter and inducible Gal4fusion proteins in T-cells. We have chosen SupT1 cells, a T-cell lineeasily infected by HIV-1 and broadly utilized in HIV-1 studies. Viralparticles were produced as described in Methods. When SupT1 cells wereinfected with lentiviral particles containing the reporter vector alone,no detectable eGFP expression was observed, and similar results wereobtained with virus encoding inducible Gal4 alone (see Figures).Importantly however, when cells were co-infected with virus producedfrom the reporter (pH-5xUAS-eGFP), rtTA (pBMN-rtTA-i-mCherry) andinducible Gal4 (pH-TRE-PRm/Gal4) encoding vectors, eGFP expression wasstill undetectable. However, when these cells were treated with 1 μg/mLDox, eGFP expression was clearly induced. This confirmed the feasibilityof engineering a Tet-off/on inducible system in T-cells to control PRexpression.

SupT1 cells were transduced with viral particles generated from thereporter, rtTA and inducible PRm/Gal4 (pH-TRE-PRm/Gal4) which resultedin similar induction of eGFP as Gal4 alone, and again only in thepresence of Dox (1 ug/ml). Finally, cells were infected with virusencoding the reporter, rtTA and inducible wild-type PR/Gal4(pH-TRE-PR/Gal4) to test the ability for this system in T-cells toindicate the levels of PR activity. In the presence of 1 μg/mL Dox, eGFPlevels were nearly undetectable. However, in the presence of both 1μg/mL Dox and 10 μM Indinavir, a large induction of eGFP expression wasobserved. This validated the ability to utilize eGFP expression as abiosensor of an active PI in SupT1 cells.

Generation and selection of monoclonal stable cell lines with thehighest responsiveness in the assay. The experiments performed withlentiviral particles were analyzed from non-clonal cell populations andcorroborated that the assay functions as expected. Nevertheless, ourgoal was to design an assay in T-cells that also had a definitive androbust readout. Therefore, it was important to purify and amplifyspecific clones from this population that possessed the lowest degree ofbackground and highest degree of eGFP expression in response to theappropriate treatment.

Cells harboring rtTA, 5xUAS-eGFP and either Gal4 or PRm/Gal4 wereactivated with 1 μg/mL of Dox. Cells harboring rtTA, 5xUASeGFP andPR/Gal4 were activated with the same concentration of Dox, however werepre-incubated with 10 μM Indinavir. All cells were then sorted 24 hourslater based on eGFP expression to enrich cells with an activatablereporter and an inducible transcription factor. One more round ofsorting was performed under identical conditions. Finally, another roundof sorting was performed seven days later to isolate cells with no eGFPexpression (i.e. cells with little to no background). This resulted in acell population that was up to 80% positive for eGFP after activationand with nearly zero background.

Finally, individual cells from these sorted populations were sorted intoa 96 well plate based on the lack of eGFP expression. Clonal cell linesobtained from this experiment were later activated under the sameconditions described above, and screened for the individual clones whichresponded as desired (minimal background and maximal eGFP activation). Aclone for each of the inducible elements (Gal4, PRm/Gal4 and PR/Gal4)was obtained. The selected clones exhibited nearly 100% activationability with nearly zero background. These clones were then expanded andfurther tested in the following experiments.

Doxycycline activation titration of the clones: In order to optimize theassay for maximal Gal4, PR/Gal4 or PRm/Gal4 induction, we analyzed theeffect of increasing levels of Dox. Cells with 0, 50, 100, 250, 500,750, 1,000, and 2,000 ng/mL Dox were either treated with 10 uM DMSO or10 uM Indinavir and analyzed 32 hours later. TRE-Gal4 cells reachedsaturation with Doxycycline at about 1,000 ng/mL whether Indinavir waspresent or not. TRE-PRm/Gal4 was surprisingly induced at lower levels,reaching saturation at around 250 ng/mL in both uninhibited andIndinavir-treated cells. Again, as with transiently transfected 293cells, TRE-PR/Gal4 cells had little to non-detectable eGFP expression atany given Dox concentration in the absence of 10 μM Indinavir. However,pre-incubation with 10 μM Indinavir showed maximal eGFP induction ataround 500 ng/mL Dox (see Figures).

Determination of optimal time point for the analysis of inhibitoreffect: To determine the optimal time point for the analysis of eGFPexpression in the presence of inhibitor, cells were activated with Doxand analyzed by flow cytometry 4, 8, 12, 16, 20, 25, 50, or 75 hourslater. Six-well plates containing 250K cells per well in 3 mL media weretreated either with DMSO alone as control, or 1 μg/mL Dox and 10 μMIndinavir. DMSO-treated cells maintained lack of fluorescence throughoutthe experiment (Figure). However, Dox-activated cells incubated withIndinavir showed an initial induction of eGFP expression at 8 hoursreaching nearly 100% by 50 hours (h).

Assay response to various PR inhibitors: Finally, in order to addressthe sensitivity of the assay to other PR inhibitors, we have analyzedthe effect of known FDA-approved inhibitors. For that purpose, cellswere incubated with DMSO alone, as described above, or with increasingconcentrations of Atazanavir, Amprenavir, Darunavir, Indinavir,Nelfinavir, Lopinavir, Ritonavir, Saquinavir and Tipranavir, all PRinhibitors, at a range including the most commonly used concentrationsin cell culture, but also including low ranges not typically active inless sensitive assays. The range chosen extended from 1 nM to 20 μM.

Darunavir and Atazanavir had the strongest effect on PR inhibition,resulting in eGFP activation at only 1 nM. Indinavir and Tipranavirshowed the lowest levels of reporter activation at low concentrations,although were also observed to induce nearly full activation at 5 μM.Interestingly, most of the tested PR inhibitors led to increasing eGFPexpression levels at increasing concentration of inhibitor throughoutthe curve, up to 20 μM, the maximal level tested, however, Lopinavir andNelfinavir resulted in significant cell death around 1 uM concentration,with significant decline in the number of eGFP positive cells as was thecase for Saquinavir at 20 μM. Overall, every inhibitor tested showedsignificant inhibitory effect that resulted in the complete activationof eGFP expression in the reporter T-cell clones.

Discussion

This exemplary assay of the invention is designed to allow for thesimple screening of novel inhibitory compound or peptide candidates in asimple flow cytometry-based platform. As such, it will allowinvestigators to perform screening of millions of candidates (throughrational or non-rational-based approaches) in a single experiment,enhancing high throughput capacity. The assay described here is intendedto greatly facilitate screening for the search of novel PR inhibitors inT-cells, one of the main natural targets of HIV infection. Importantly,we have established an assay whose elements can be easily transferred toother relevant cell types for the establishment of HIV infection and thesearch for novel PR inhibitors. These include cells such as macrophagesor dendritic cells.

The mean fluorescence intensity of eGFP in activated cells steadilyincreased as inhibited cells continued to accumulate higher levels ofeGFP, making those cells with an inhibited PR even more identifiablethan those negative for PR inhibition.

In some aspects, analysis of a time point for the effect of an inhibitormay be critical. Thus, choosing a time-point that is average for all isonly an estimation for the search of unknown inhibitors.

Methods

Cloning and Vector Construction: Gal4, PR/Gal4 and PRm/Gal4 sequenceswere amplified by PCR for the production of the transient expressionvectors pcDNA-Gal4, pcDNA-PR/Gal4 and pcDNA-PRm/Gal4 respectively. Forthat purpose the constructs pMA236, pHP236 and pHP236m (Murray et al.,1993) were used as template together with the Gal4 forward primer withextending HindIII and NotI sitesACGCACGCAAGCTTGCGGCCGCCCACCATGAAGCTACTGTCTTCTATC (SEQ ID NO:6) and theGal4 reverse primer with extending SalI siteATAGCTGCGTGCGTGCGTGTCGACTTACTCTTTTTTTGGGTTTGG (SEQ ID NO:7).

PCR products were then digested with HindIII and SalI and ligated intopcDNA™3.1/Zeo (Invitrogen, Carlsbad Calif.). pFR-eGFP was kindlyprovided by Rainer de Martin (University of Vienna). In short, pFR-eGFPwas created by adapting the pFR-Luc vector (Stratagene, San Diego,Calif.). The firefly luciferase gene was removed and replaced with eGFP.

For the construction of the inducible pH-TRE, pH-TRE-eGFP pH-TRE-Gal4,pH-TRE-PR/Gal4, pH-TRE-PRm/Gal4 vectors, a 7× tet-responsive element(TRE) was amplified from the pTRE-tight (Clontech) with the forwardprimer with extending NruI site AGCTAGCTAGCTTCGCGACACGAGGCCCTTTCGTCTTCA(SEQ ID NO:8) and a reverse primer to complementary to the PolyA signalwith an extending BsrGI site CATTTTTTTCACTGCCTCGAGTGTACAAGCTAGCTAGCT(SEQ ID NO:9).

The PCR product was digested with NruI/BsrGI and cloned into thepH-CMV-eGFP vector, (generously provided by Gary Nolan, StanfordUniversity, CA) to replace the original CMV-eGFP cassette. eGFP was thenamplified with a forward primer containing an extending BamHI site, anda reverse primer with an extending NheI site. The eGFP insert was thencut and ligated within the multiple cloning site of pH-TRE. The forwardGal4 primer with an extending BamHI siteATGCATGCGGATCCACCATGAAGCTACTGTCTTCTATC (SEQ ID NO:10). and the reverseprimer with an extending NheI site GCATGCATGCTAGCTTACTCTTTTTTTGGGTTTGG(SEQ ID NO:11). were used to amplify the Gal4-based cassettes frompcDNA3.1-Gal4, pcDNA3.1-PR/Gal4, and pcDNA3.1-PRm/Gal4 and insert theminto pH-TRE digested with BamHI/NheI.

pBMN-i-mCherry was constructed by amplifying mCherry from pmCherry-C1(Clontech) with the forward primer with extending NcoI siteATCGATGGATCCCCACCATGGTGAGCAAGGGCGAGGAG (SEQ ID NO:12). and reverseprimer with extending XhoI site: ATGGACGAGCTGTACAAGTAACTCGAGGATCGATC(SEQ ID NO:13), and inserting it into partially digested pBMN-i-eGFP(Gary Nolan, Stanford University) with NcoI/SalI. pBMN-i-mCherry-rtTAwas then constructed by removing rtTA from the vector Tet-On® (Clontech)with EcoRI/BamHI and cloning it into pBluescript-SK (Invitrogen,Carlsbad Calif.). It was then removed with EcoRI/XhoI and ligated intopBMN-i-mCherry.

pH-5XUAS-eGFP was constructed by digesting pFR-eGFP with MfeI/BsrGI andligating the 5XUAS-eGFP insert into pH-CMV-eGFP digested with MfeI/BsrGIto replace the CMV-eGFP cassette.

Transfections: 293T cells were transfected as follows. 15 μl of 2 mg/mLPolyethylenimine linear 25 kD (Polysciences, Inc.) were added to 125 μlof DMEM in a 1.8 ml Eppendorf tube. 3 μg of each DNA was added drop-wiseto each tube. Tubes were mixed and incubated for 20 min at RT. Thismixture was then added drop-wise to 293T cells in a 10 cm plate at˜60-75% confluence. Cells were then analyzed by fluorescence microscopyand/or flow cytometry 24 hrs post transfection.

Production of Viral Particles for Retroviral Transduction: For theproduction of MLV based virus (pBMN-i-mCherry-rtTA virus), Phoenix GPcells (Nolan Lab, Stanford University, CA) at 50-60% confluence weretransfected with 3 ug of the packaging vector (pBMN-i-mCherry-rtTA) and3 μg of pCI-VSVg envelope vector. Media was changed after 24 hoursleaving 6 mL of media (DMEM with 10% FCS, Pen-Strep, L-Glutamine) in a10 cm plate. At 48 hours, viral supernatant was collected, filtered with0.45 micron PTFE filters (Pall Corporation) and frozen at −80° C. in 1mL aliquots and frozen at −80° C.

For the production of HIV based virus (pH vectors), 293T cells at 50-60%confluency were transfected with 3 μg packaging vector (pH vectors), 2ug pCI-VSVg, VPR encoding vector, and 3 μg pCMVΔ8.2 (Didier Trono, EPFL,Switzerland). Media was changed at 24 hours leaving 6 mL fresh media(DMEM with 10% FCS, Pen-Strep, L-glutamine) Supernatant was collected at48 hrs, filtered and collected as described above.

Infections: A 2 ml reaction containing 4 μl of 5 μg/mL Polybrene(Hexadimethrene Bromide, Sigma), 500K SupT1 cells in RPMI (10% FCS,L-glutamine, Pen-strep) and 250 μl frozen viral stocks was mixed, addedto wells in 6-well plates, and spun in a hanging bucket rotors BectonDickinson centrifuge at 1500 RPM, for 120′ at 32° C. Cells werere-suspended and placed in a 37° C. incubator for the analysis ofexpression at least 72 hours post-infection.

Fluorescence Microscopy for Analysis of Expression: Cells were analyzedby fluorescence microscopy on a Zeiss Observer D1 microscope with a 50×lens and 40HMC filter connected to an AxioCam MRm camera, and analyzedon Axio-Vision software. The length of exposure for fluorescent channelswas based on the exposure for the Gal4 only controls. This length wasthen kept constant for the exposures of all of the other samples.

Flow Cytometry and Sorting: Flow Cytometry was performed on a BDFACSAria with 488 nm and 633 nm lasers. Data was collected on FACSDiva6.1.1 software and then exported to FlowJo. eGFP expression was detectedin the FITC channel and mCherry expression was detected on thePE-Texas-Red channel. Cells were first gated for size and granularity(FSC-A vs SSC-A) followed by doublet gating (FSC-A vs FSC-W and SSC-Avs. SSC-W). Sorted populations were gated on PE-TexasRed vs FITC plot.250K cells were collected for each sample into 0.5 mL fetal calf serum(FCS) and 1 mL RPMI. Cells were then spun down at 1500 RPM andresuspended in fresh media in 6-well plates. Cells were allowed to growfor at least seven days to allow for expansion and loss of previouslyactivated eGFP expression.

Figure Legends Example 2

FIG. 19 illustrates an overview of the assay: FIG. 19A. Wild type Gal4as control, no Dox. Without the presence of Dox, rtTA can not bind tothe tet-responsive element (TRE) and as a result, there is no inductionof Gal4 expression. Consequently, there is no eGFP expressed from thereporter construct. FIG. 19B. In the presence of Dox, Gal4 expression isinduced. Gal4 then binds to the 5xUAS of the reporter gene and activateseGFP expression. FIG. 19C. The same system with the PR/Gal4 fusion. Inthe presence of Dox PR/Gal4 is expressed; however, its catalyticactivity results in the separation of the Gal4 domains, and thus thereis no yield of eGFP. FIG. 19D. The same scenario as in C but in thepresence of a PR inhibitor. PR/Gal4 fusion with Gal4 is retained,resulting in the induction of eGFP expression. Same results are expectedif PR is mutated, with no additional need of PR inhibitors.

FIG. 20 illustrates a transient expression of the assay components inHEK293T cells. FIG. 20A schematically illustrates exemplary constructsused for transient expression of the assay elements. FIG. 20B. TopPanel: Fluorescence microscopy of eGFP expression in HEK293T cells 24hours post transfection with the reporter vector (pFR), orco-transfection with the reporter vector plus either the Gal4, PR/Gal4,or PRm/Gal4 vectors. Bottom panel: HEK293T cells were analyzed by flowcytometry at 24 hours post transfection with the same conditions toppanel. Gates were drawn to determine the ability to identify cells withan active PI. FIG. 20C. Quantification of eGFP expression in HEK293Ttransfected with various assay elements. The numbers of cells undervarious conditions were quantified for eGFP levels indicative of PRinhibition. Results shown are an average of 3 experiments.

FIG. 21 illustrates generation of a monoclonal T-cell line stablyexpressing inducible assay elements. FIG. 21A (upper left) schematicallyillustrates exemplary constructs utilized to generate infectiousparticles for the transduction of SupT1 cells with the various assayelements. FIG. 21B (right) illustrates data from a cell sorting assaycomprising: a previously generated stable SupT1 cell line expressingrtTA and mCherry was infected with the reporter virus (generated frompH-5xUAS-eGFP), or co-infected with the reporter virus plus a virusencoding an inducible assay element (generated from pH-TRE-Gal4,pH-TRE-PR/Gal4, or pH-TRE-PRm/Gal4). Cells were then activated witheither 1 μg/mL of Dox alone, or were pre-incubated for 5 minutes with 10μM Indinavir following activation with 1 μg/mL Dox. Initial infectionswith low yields (left column) were then subjected to several rounds ofsorting on a BD FACSAria to obtain a cell population with a higher assayresponse (middle column). Finally, single SupT1 cells from the purifiedpopulation were sorted into 96 well plates and grown as a monoclonalpopulation and then tested for optimal responses to assay induction ofeGFP under appropriate conditions. FIG. 21C (lower left) illustratesimages of fluorescence microscopy of SupT1 clones expressing the assayelements. Cells expressing various elements of the assay were treated asindicated with DMSO, DMSO+1 μg/mL Dox, or 1 μl/mL Dox+10 μM Indinavirand analyzed 24 hours later by fluorescence microscopy for mCherry andeGFP expression. D. Quantification of eGFP expression of clonal SupT1cells treated with DMSO, DMSO+1 μg/mL Dox, or 1 μg/mL Dox+10 μMIndinavir.

FIG. 22 graphically illustrates data determining the optimal conditionsfor activating the assay to screen for PR inhibition. FIG. 22Agraphically illustrates a Doxycycline titration. Clonal SupT1 cellsharboring an inducible Gal4, PR/Gal4 or PRm/Gal4 were pre-incubated in a96 well plate with either DMSO or 10 μM Indinavir and then either leftuntreated, or activated with 50, 100, 200, 500, 1000, or 20000 ng/mL ofDox. Cells were analyzed by flow cytometry and gated to determine thenumber of cells positive for eGFP expression. FIG. 22B graphicallyillustrates a time course of eGFP Induction in response to Doxycylineactivation in the presence of DMSO or a PI. SupT1 clones harboring Gal4,PR/Gal4, or PRm/Gal4 were pre-incubated in a 96 well plate with eitherDMSO or 10 μM Indinavir and then activated with 1 μg/mL Dox. Cells wereanalyzed at either 4, 8, 12, 16, 20, 25, 50 or 75 hours by flowcytometry and gated for eGFP positive expression.

FIG. 23 graphically illustrates data from an assay response to existingPI's. The selected SupT1 clone expressing rtTA and mCherry, the5xUAS-eGFP reporter and an inducible PR/Gal4 were activated with 1 μg/mLand incubated with various concentrations (1, 10, 50, 100, 500, 1000,5000, 10000 or 20000 ng/mL) of an FDA approved PI: Either Amprenavir,Atazanavir, Darunavir, Indinavir, Lopinavir, Nelfinavir, Ritonavir,Saquinavir, or Tipranavir. Cells were then analyzed 50 hrs later foreGFP expression as determined by flow cytometry on a BD FACSAria.

Example 3 Exemplary Assays and Multiplexed Systems—ConditionalProtease/Gal4 Fusion-Based Systems

In alternative embodiments, the invention provides compositions andassays to screen for both protease inhibitors, including viral proteaseinhibitors such as PR, that can be used e.g., as anti-viral oranti-retroviral therapy against e.g., HIV-1, or AIDS, which remains adevastating disease. The invention provides assays for identifying noveldrugs and targets in the fight against HIV and other diseases. Theinvention provides assays for identifying novel drugs not having theside-effects of existing drugs. The invention provides assays foridentifying novel drugs effective against strains resistant to knowndrugs, e.g., because of the high rate of HIV mutation.

In alternative embodiments, the invention provides protease assays,e.g., HIV Protease (PR) assays, for the detection of novel proteaseinhibitors (PI's), e.g., HIV protease inhibitors, in vivo, or in a cellsuch as a mammalian cell, a T cell, a bacteria or a yeast, or in vitro.In alternative embodiments, the invention's assays screen for PI'swithin T-cells, thus allowing a search for inhibitory compounds within arealistic cellular environment and producing more reliable hits.Additionally, in this embodiment the screening can concurrently revealthe toxicity level of drug candidates on T-cells, ruling out lethalhits.

We have engineered a clonal T-cell line with a Doxycycline induciblePR-Gal4 fusion and a Green Fluorescent Protein (GFP), or eGFP, reporterof its activity. Thus, in this exemplary assay of the invention, eGFPacts as a biosensor of PR activity, making it ideal for flowcytometry-based screening. Clones with the highest sensitivity, androbust, reliable and reproducible reporter activity were tested fortheir ability to detect the presence of a potent PI. The selected clonesexhibit eGFP expression in nearly 100% of the population with theaddition of every FDA-approved inhibitor tested, with sensitivitiesranging down to nanomolar concentrations. This exemplary assay/platformof the invention is a High Throughput Screening assay for PIs that canbe performed in T-cells and other mammalian cells, and can facilitatethe search for novel peptide- and chemical-compound-based PIs.

FIG. 24 schematically illustrates an exemplary assay of the invention, aconditional Protease/Gal4 fusion-based system, where GFP is activatedonly in the presence of a Protease Inhibitor:

-   -   FIG. 24 A schematically illustrates: No doxycycline, Gal4 (DB        and TA domains) cannot be expressed.    -   FIG. 24 B schematically illustrates: In the presence of        doxycycline: D, rtTA binds to the tet-responsive element (TRE)        and induces Gal4 expression resulting in the activation of GFP        expression.    -   FIG. 24 C schematically illustrates: Protease/Gal4 is expressed;        however, its catalytic activity results in the separation of the        Gal4 domains, resulting in the lack of GFP expression.    -   FIG. 24 D schematically illustrates: In the presence of a        protease inhibitor (PI), the PR/Gal4 fusion remains intact,        resulting in the induction of GFP expression. The same result is        expected with an inactive mutant PR.

Transient Assay in Mammalian Cells:

FIG. 25 schematically illustrates exemplary plasmids of the inventionfor transfection in HEK293T cells; PRm=inactive mutant (D25A). theplasmids are named, from top to bottom of the figure: “pFR-GFP”;“pGal4”; “pPRm/Gal4”; and “pPR/Gal4”.

FIG. 26 illustrates data from a fluorescent cell sorting assay, a FACS,showing that GFP is expressed only with the addition of an active PI.

FIG. 27 graphically summarizes the data analysis for 24 h posttransfection: FIG. 27A. Fluorescence microscopy; FIG. 27B. Flowcytometry; FIG. 27C Quantification of Flow data; PI=10 μM Indinavir.

FIG. 28 illustrates data: the y axis is: % GFP+ cells; the x axis is+FRGFP; for each of the paired columns the left column is control andthe right column is with inhibitor; the first lane (no columns) is mockrun; the second column pair is negative control; the third column pairis “pGal4”; the fourth column pair is “pPRm/Gal4”; and the fifth columnpair is “pPR/Gal4”.

Stable T cell clones:

FIG. 29 illustrates constructs used for the generation of retroviralparticles.

FIG. 30 illustrates plasmids for production of retroviral infectiousparticles.

FIG. 31 illustrates data from a cell sorting assay where clones werescreened for the highest responsiveness to Dox and PI; where the Gal 4row shows that Tet inducible activation is very tight; the pPRm rowshows that the mutant is inactive; the PR row shows that PR/Gal4 clonesexhibit=90% activation with <1% background. Dox=1 μg/mL Doxycycline;PI=1004 Indinavir.

FIG. 32 graphically illustrates GFP expression in selected clones, wherethe data demonstrates that stable T-cell clones robustly report PRInhibition.

Optimization of PI Screening Conditions:

FIG. 33 graphically illustrates Doxycycline Titration—pre-incubation ofclonal SupT1 cells with DMSO or 10 μM PI (Indinavir), where the datademonstrates that activation is saturated around 1 μg/mL.

FIG. 34 graphically illustrates the Time Kinetics of the assay in96-well plates: Pre-incubation of clonal SupT1 cells with 10 μM PI(Indinavir)—control is no Dox with test=Activation at 1 mg/mL Dox, wherethe data demonstrates that activation of clones reaches max around 48hrs.

Assay Response to FDA-Approved PIs:

FIG. 35 graphically illustrates data from incubating clonal T-cell lineswith various concentrations of PI's: activation with 1 μg/mL Dox;analyzed 50 hrs later by flow cytometry, where the data demonstratesthat at the nanomolar range, the clonal T-cell line shows PR inhibitionof every FDA-approved PI.

Example 4 Cell-Based Assays for the Identification of Compositions thatInhibit Envelop Processing

In alternative embodiments, the assay screens ER/Golgi-localized randompeptide libraries for anti-virals and/or inhibitors of furins or similarassays.

FIG. 36 schematically illustrates the HIV-1 genome and proteome, and therole of furin, PC-1 and similar host peptidases—the enzymes targeted forinhibition by assays of this invention. Post translational processing ofthe viral proteome includes cleavage by both viral and host cellproteases; and assays of the invention can identify inhibitors of bothviral and host cell proteases. Viral proteins are processed by the HIV-1protease with the exception of gp120/gp41.

FIG. 37 schematically illustrates an exemplary assay of the invention.FIG. 37 Right panel: when furin, or similar proteinases are blocked orinhibited, the scaffold protein is not cleaved and thus is retained inthe ER. FIG. 37 Left panel: If cleaved, the scaffold travels to the cellsurface leaving the KDEL sequence behind. This can recognized by flowcytometry, e.g., a FACS, or by using a high throughput screen, or usingmicroscope visualization, which can be automated. The cleaved scaffoldwill travel from the ER through the trans-Golgi network and to the cellsurface, allowing recognition by flow cytometry. Right panel: Thescaffold will be retained in the ER through KDEL when cellularpeptidases such as Furin are blocked or inhibited, e.g., by peptides orsmall molecules identified using assays of this invention.

FIG. 38 schematically illustrates constructs for assays of theinvention: two retroviral vectors are illustrated: Top: Scaffoldconstruct: the ER-signal sequence followed by the FLAG tag for detectionby flow cytometry are fused to two trans-membrane domains (TMs) from theCCR5 receptor. Following the TMs, the scaffold contains the gp120/41boundary that includes the cleavage site. The sequence KRRVVQREKRAVGIGAL(SEQ ID NO:18) (which are residues two (2) to eighteen (18) of SEQ IDNO:17, or AKRRVVQREKRAVGIGALF) is taken from the HXB2 HIV-1 strain.Importantly, the KDEL (SEQ ID NO:1) ER-retention sequence, at theC-terminus of the construct, will allow localization in the ER lumenthrough KDEL receptors. Bottom: the back-bone of the peptide library isfused to the KDEL sequence and linker to allow flexibility within the ERlumen. In one embodiment, while the scaffold can be detected byfluorescence (mCherry), the library can be selected with blasticidin.

FIG. 39 graphically illustrates flow cytometry data from a FLAGdetection assay of the invention: SupT1 cells were stained withanti-FLAG antibodies and detected by flow cytometry. Left: Control naïvecells. Right: Cells expressing FLAG-tagged CCR5.

FIG. 40 schematically illustrates an exemplary screening process for anassay of the invention: In this embodiment, SupT1 cells expressing thescaffold (mCherry positive) are transduced with the retroviral randompeptide library localized to the ER-trans-Golgi apparatus. Cellsexpressing peptides (blasticidin resistant) are selected. In oneembodiment, cells are analyzed by flow cytometry to detect the loss ofFLAG cell-surface expression. In one embodiment, these cells are sortedand amplified. In one embodiment, the peptide sequence are rescued bygenomic PCR and analyzed for its effects on envelope processing.

FIG. 41 schematically illustrates construction of a random peptidelibrary used in an alternative embodiment of the invention. In oneembodiment, preparation of random peptide library insert utilizes an NNKmotif to minimize stop codons and preserve complexity of the peptidelibrary. CCACCATG(NNK)nTGA is (SEQ ID NO:19); and in one embodiment,these sequences contain a Kozak sequence (or Kozak consensus sequence,Kozak consensus or Kozak sequence, is a sequence which occurs oneukaryotic mRNA and plays a major role in the initiation of thetranslation process).

FIG. 42A illustrates exemplary library inserts to corroborate quality,clones were sequenced to confirm their randomness; FIG. 42B illustratesan electrophoresis analysis.

Exemplary assays of the invention are constructed in T-cells tofacilitate the identification of novel drugs targeting viral envelopeprocessing. Assays of the invention represent a novel way to monitorprocessing by ER-trans-Golgi resident peptidases. As such, assays of theinvention enable both the search for novel inhibitors, and further helpelucidate mechanisms of protein cellular membrane transport. Inalternative embodiments, assays of the invention are used to screenrandom peptide libraries and/or small molecule libraries for thediscovery of possible novel inhibitors that will target gp160 processingrather than Furin activity.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A cell-based method for monitoring the activity of a protease, aviral protease, or an HIV-1 protease (PR), comprising: (1) (a) providinga nucleic acid encoding a scaffold protein operatively linked to atranscriptional regulatory unit, wherein the scaffold protein comprises:(i) an amino acid motif or subsequence susceptible to cleavage by theprotease, viral protease or HIV-1 protease (PR), under physiologic (cellculture) conditions; (ii) a transmembrane domain; (iii) a signalsequence or any amino acid motif that places the scaffold protein on theextracellular surface of the cell; and (iv) a detectable moiety, whereinthe amino acid motif or subsequence susceptible to cleavage by theprotease, viral protease or HIV-1 protease (PR) is positioned within thescaffold protein such that when the detectable moiety is cleaved awayfrom (off from) the scaffold protein by the protease, viral protease orHIV-1 protease (PR) the remaining subsequence of scaffold protein on theextracellular surface of the cell lacks the detectable moiety; (b)providing a nucleic acid encoding the protease, viral protease or HIV-1protease (PR) operatively linked to a transcriptional regulatory unit,or a cell that expresses a heterologous or endogenous protease, viralprotease or HIV-1 protease (PR); (c) inserting (transfecting) thenucleic acid of (a) and (b) into the cell if the cell does not alreadyexpress a heterologous or endogenous protease, viral protease or HIV-1protease (PR); (d) co-expressing the nucleic acid of (a) and (b) in thecell, or expressing the nucleic acid of (a) in the cell if the cellalready expresses a heterologous or endogenous protease, viral proteaseor HIV-1 protease (PR); and (e) determining whether the scaffold proteincomprising the detectable moiety is expressed on the extracellularsurface of the cell, wherein an intact scaffold protein comprising thedetectable moiety is expressed on the extracellular surface of the cellwhen the protease, viral protease or HIV-1 protease (PR) is notenzymatically active, and an intact scaffold protein is not or issubstantially less expressed on the extracellular surface of the cellwhen the protease, viral protease or HIV-1 protease (PR) isenzymatically active (the detectable moiety is cleaved off by theprotease, viral protease or HIV-1 protease (PR)); (2) the method of (1),wherein the scaffold protein further comprises an endoplasmic reticulum(ER) retention motif or a KDEL (SEQ ID NO:1) motif, wherein the ERretention motif or KDEL (SEQ ID NO:1) motif is positioned in thescaffold protein such that when PR is active the scaffold will beseparated into two pieces, leaving the ER retention motif-comprising orKDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ERand freeing the detectable moiety-comprising portion to the cell'sextracellular membrane, and if PR is blocked or inactive, the entirescaffold polypeptide will be retained in the ER, and as a consequencewill not be detected on the cell's extracellular surface; or (3) themethod of (1), wherein the scaffold protein further comprises a p2/p7recognition site imbedded in the cytoplasmic loop of the scaffold. 2.The method of claim 1, further comprising: (1) screening for aninhibitor of a protease, a viral protease or an HIV-1 protease (PR) by:(a) providing a compound to be screened as an inhibitor of a protease,viral protease or HIV-1 protease (PR), or providing a nucleic acid to bescreened as encoding an inhibitor of a protease, viral protease or HIV-1protease (PR); (b) contacting a plurality of the cells with the compoundor nucleic acid of (a) either before, during and/or after theco-expressing the nucleic acid of claim 1(a) and claim 1(b) in the cell;and (c) determining whether the scaffold protein comprising thedetectable moiety is expressed on the extracellular surface of the cell,wherein an intact scaffold protein comprising the detectable moiety isexpressed on the extracellular surface of the cell when the protease,viral protease or HIV-1 protease (PR) is inhibited by: the compound, acomposition encoded by the nucleic acid, or a compound present in thecell only because the nucleic acid was expressed, and an intact scaffoldprotein is not or is substantially less expressed on the extracellularsurface of the cell when the protease, viral protease or HIV-1 protease(PR) is enzymatically active (the detectable moiety is cleaved off bythe protease, viral protease or HIV-1 protease (PR)) and the enzymaticactivity of the protease, viral protease or HIV-1 protease (PR) is notsignificantly inhibited by: the compound, a composition encoded by thenucleic acid, or a compound present in the cell only because the nucleicacid was expressed (2) the method of (1), further comprising running anegative control comprising dividing the plurality of the cellsco-expressing the nucleic acid of (a) and (b) in the cell and not addingthe compound to be screened as an inhibitor to one of the divided cellsamples; or (3) the method of (1) or (2), further comprising running apositive control comprising dividing the plurality of the cellsco-expressing the nucleic acid of (a) and (b) in the cell and adding aknown inhibitor of the protease, viral protease or HIV-1 protease (PR)to one of the divided cell samples. 3-4. (canceled)
 5. The method ofclaim 1, wherein: (a) the amino acid motif or subsequence susceptible tocleavage by the HIV-1 protease (PR) under physiologic (cell culture)conditions comprises SEQ ID NO:3 or SEQ ID NO:4; (b) the HIV-1 protease(PR) comprises SEQ ID NO:5 or SEQ ID NO:6; (c) the transcriptionalregulatory unit comprises a promoter, an inducible promoter or aconstitutive promoter; (d) the cell is a mammalian cell, a monkey cellor a human cell; (e) the scaffold proteins comprise all or part of amouse Lyt2 or a human CD8 polypeptide; (f) the detectable moietycomprises an epitope for an antibody, or a FLAG tag; (g) the detectablemoiety is detected or measured on the extracellular surface of the cellby a high throughput screen, a flow cytometry or microscopevisualization; (h) the compound to be screened as an inhibitor of theprotease, viral protease or HIV-1 protease (PR) comprises a smallmolecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, apolysaccharide or a lipid; or (i) the compound to be screened as aninhibitor of the protease, viral protease or HIV-1 protease (PR) is amember of a library of compounds to be screened, or a member of a randompeptide library or a chemical compound library. 6-13. (canceled)
 14. Acell-based method for monitoring the activity of a protease comprising:(1) (a) providing a nucleic acid encoding a scaffold protein operativelylinked to a transcriptional regulatory unit, wherein the scaffoldprotein comprises: (i) an amino acid motif or subsequence susceptible tocleavage by the protease under physiologic (cell culture) conditions;(ii) a transmembrane domain; (iii) a signal sequence or any amino acidmotif that places the scaffold protein on the extracellular surface ofthe cell; and (iv) a detectable moiety, wherein the amino acid motif orsubsequence susceptible to cleavage by the protease is positioned withinthe scaffold protein such that when the detectable moiety is cleavageaway from (off from) the scaffold protein by the protease the remainingsubsequence of scaffold protein on the extracellular surface of the celllacks the detectable moiety; (b) providing a nucleic acid encoding theprotease operatively linked to a transcriptional regulatory unit, or acell that expresses a heterologous or endogenous protease; (c) inserting(transfecting) the nucleic acid of (a) and (b) into the cell if the celldoes not already express a heterologous or endogenous protease; (d)co-expressing the nucleic acid of (a) and (b) in the cell, or expressingthe nucleic acid of (a) in the cell if the cell already expresses aheterologous or endogenous protease; and (e) determining whether thescaffold protein comprising the detectable moiety is expressed on theextracellular surface of the cell, wherein an intact scaffold proteincomprising the detectable moiety is expressed on the extracellularsurface of the cell when the protease is not enzymatically active, andan intact scaffold protein is not or is substantially less expressed onthe extracellular surface of the cell when the protease is enzymaticallyactive (the detectable moiety is cleaved off by the protease); or (2)the method of (1), wherein the scaffold protein further comprises anendoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1)motif, wherein the ER retention motif or KDEL (SEQ ID NO:1) motif ispositioned in the scaffold protein such that when PR is active thescaffold will be separated into two pieces, leaving the ER retentionmotif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of thepolypeptide in the ER and freeing the detectable moiety-comprisingportion to the cell's extracellular membrane, and if PR is blocked orinactive, the entire scaffold polypeptide will be retained in the ER,and as a consequence will not be detected on the cell's extracellularsurface.
 15. The method of claim 14, further comprising screening for aninhibitor of a protease by: (1)(a) providing a compound to be screenedas an inhibitor of a protease, or providing a nucleic acid to bescreened as encoding an inhibitor of a protease; (b) contacting aplurality of the cells with the compound or nucleic acid of claim 14(a)either before, during and/or after the co-expressing the nucleic acid ofclaim 14(a) and claim 14(b) in the cell; and (c) determining whether thescaffold protein comprising the detectable moiety is expressed on theextracellular surface of the cell, wherein an intact scaffold proteincomprising the detectable moiety is expressed on the extracellularsurface of the cell when the protease is inhibited by: the compound, acomposition encoded by the nucleic acid, or a compound present in thecell only because the nucleic acid was expressed, and an intact scaffoldprotein is not or is substantially less expressed on the extracellularsurface of the cell when the protease is enzymatically active (thedetectable moiety is cleaved off by the protease) and the enzymaticactivity of the protease is not significantly inhibited by: thecompound, a composition encoded by the nucleic acid, or a compoundpresent in the cell only because the nucleic acid was expressed; (2) themethod of (1), further comprising running a negative control comprisingdividing the plurality of the cells co-expressing the nucleic acid of(a) and (b) in the cell and not adding the compound to be screened as aninhibitor to one of the divided cell samples; (3) the method of (1) or(2), further comprising running a positive control comprising dividingthe plurality of the cells co-expressing the nucleic acid of (a) and (b)in the cell and adding a known inhibitor of the protease to one of thedivided cell samples; (4) the method of any of (1) to (3), wherein thetranscriptional regulatory unit comprises a promoter, or an induciblepromoter or a constitutive promoter; (5) the method of any of (1) to(4), wherein the cell is a mammalian cell, a monkey cell or a humancell; (6) the method of any of (1) to (5), wherein the scaffold proteinscomprise all or part of a mouse Lyt2 or a human CD8 polypeptide; (7) themethod of any of (1) to (6), wherein the detectable moiety comprises anepitope for an antibody, or a FLAG tag; (8) the method of any of (1) to(7), wherein the detectable moiety is detected or measured on theextracellular surface of the cell by a high throughput screen, a flowcytometry or microscope visualization; (9) the method of any of (1) to(8), wherein the compound to be screened as an inhibitor of proteasecomprises a small molecule, a nucleic acid, a polypeptide or peptide, apeptidomimetic, a polysaccharide or a lipid, or the compound to bescreened as an inhibitor of protease is a member of a library ofcompounds to be screened, or a member of a random peptide library or achemical compound library; or (10) the method of any of (1) to (9),wherein the protease is an HIV-1 protease (PR), or the protease is aviral, a microbial or a mammalian protease. 16-28. (canceled)
 29. Acell-based method for monitoring the activity of a cell's ER and/ortrans-Golgi network comprising: (1) (a) providing a nucleic acidencoding a scaffold protein operatively linked to a transcriptionalregulatory unit, wherein the scaffold protein comprises: (i) atransmembrane domain; (ii) a signal sequence or any amino acid motifthat places the scaffold protein on the extracellular surface of thecell; and (iii) a detectable moiety; (b) inserting (transfecting) thescaffold protein-encoding nucleic acid of (a) into the cell; (d)expressing the nucleic acid of (a); and (e) determining whether thescaffold protein comprising the detectable moiety is expressed on theextracellular surface of the cell, wherein the scaffold protein isexpressed on the extracellular surface of the cell when the activity ofthe cell's ER and trans-Golgi network is functioning; or (2) the methodof (1), wherein the scaffold protein further comprises an endoplasmicreticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif, whereinthe ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in thescaffold protein such that when PR is active the scaffold will beseparated into two pieces, leaving the ER retention motif-comprising orKDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ERand freeing the detectable moiety-comprising portion to the cell'sextracellular membrane, and if PR is blocked or inactive, the entirescaffold polypeptide will be retained in the ER, and as a consequencewill not be detected on the cell's extracellular surface.
 30. The methodof claim 29, further comprising screening for an inhibitor of the cell'sER and trans-Golgi network by: (1) (a) providing a compound or nucleicacid to be screened as an inhibitor of the cell's ER and trans-Golginetwork; (b) contacting a plurality of the cells with the compound ornucleic acid of (a) either before, during and/or after the co-expressingthe nucleic acid of claim 29(a) in the cell; and (c) determining whetherthe scaffold protein comprising the detectable moiety is expressed onthe extracellular surface of the cell, wherein an intact scaffoldprotein comprising the detectable moiety is expressed (or issubstantially expressed) on the extracellular surface of the cell whenthe cell's ER and trans-Golgi network is not inhibited (2) the method of(1), further comprising running a negative control comprising dividingthe plurality of the cells co-expressing the nucleic acid of claim 29(a)in the cell and not adding the compound to be screened as an inhibitorto one of the divided cell samples; (3) the method of (1) or (2),further comprising running a positive control comprising dividing theplurality of the cells co-expressing the nucleic acid of claim 29(a) inthe cell and adding a known inhibitor of the cell's ER and/ortrans-Golgi network to one of the divided cell samples; (4) the methodof any of (1) to (3), wherein the transcriptional regulatory unitcomprises a promoter, an inducible promoter or a constitutive promoter;(5) the method of any of (1) to (4), wherein the cell is a mammaliancell, a monkey cell or a human cell; (6) the method of any of (1) to(5), wherein the scaffold proteins comprise all or part of a mouse Lyt2or a human CD8 polypeptide; (7) the method of any of (1) to (6), whereinthe detectable moiety comprises an epitope for an antibody, or a FLAGtag; (8) the method of any of (1) to (7), wherein the detectable moietyis detected or measured on the extracellular surface of the cell by ahigh throughput screen, a flow cytometry or microscope visualization;(9) the method of any of (1) to (8), wherein the compound to be screenedas an inhibitor of protease comprises a small molecule, a nucleic acid,a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid;(10) the method of any of (1) to (9), wherein the compound to bescreened as an inhibitor of protease is a member of a library ofcompounds to be screened, or a member of a random peptide library or achemical compound library; or (11) the method of any of (1) to (10),wherein the protease is an HIV-1 protease (PR), or the protease is aviral, a microbial or a mammalian protease. 31-41. (canceled)
 42. Anisolated, recombinant or synthetic nucleic acid encoding a scaffoldprotein operatively linked to a transcriptional regulatory unit, whereinthe scaffold protein comprises: (1) (a) (i) an amino acid motif orsubsequence susceptible to cleavage by a protease under physiologic(cell culture) conditions; (ii) a transmembrane domain; (iii) a signalsequence or any amino acid motif that places the scaffold protein on theextracellular surface of the cell; and (iv) a detectable moiety; or (b)the nucleic acid of (a), wherein the scaffold protein further comprisesan endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1)motif, wherein the ER retention motif or KDEL (SEQ ID NO:1) motif ispositioned in the scaffold protein such that when PR is active thescaffold will be separated into two pieces, leaving the ER retentionmotif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of thepolypeptide in the ER and freeing the detectable moiety-comprisingportion to the cell's extracellular membrane, and if PR is blocked orinactive, the entire scaffold polypeptide will be retained in the ER,and as a consequence will not be detected on the cell's extracellularsurface; (2) the isolated, recombinant or synthetic nucleic acid of (1),wherein the protease is an HIV-1 protease (PR), or the protease is aviral, a microbial or a mammalian protease; (3) the isolated,recombinant or synthetic nucleic acid of (1) or (2), wherein thescaffold protein comprise all or part of a mouse Lyt2 or a human CD8polypeptide; or (4) the isolated, recombinant or synthetic nucleic acidof any of (1) to (3), wherein the detectable moiety comprises an epitopefor an antibody, or a FLAG tag. 43-90. (canceled)